Oligomeric compounds for the modulation of hif-1a expression

ABSTRACT

Oligonucleotides directed against the hypoxia-inducible factor-1α (HIF-1α) gene are provided for modulating the expression of HIF-1α. The compositions comprise oligonucleotides, particularly antisense oligonucleotides, targeted to nucleic acids encoding the HIF-1α. Methods of using these compounds for modulation of HIF-1α expression and for the treatment of diseases associated with the hypoxia-inducible factor-1α are provided. Examples of diseases are cancer and pre-eclampsia. The oligonucleotides may be composed of deoxyribonucleosides, a nucleic acid analogue, or Locked Nucleic Acid (LNA) or a combination thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Provisionalapplication Ser. No. 60/370,126, filed Apr. 5, 2002 of which applicationis fully incorporated herein by reference.

FIELD OF THE INVENTION

The present invention provides compositions and methods for modulatingthe expression of HIF-1α. In particular, this invention relates tooligomeric compounds and preferred such compounds are oligonucleotides,which are specifically hybridisable with nucleic acids encoding HIF-1α.The oligonucleotide compounds have been shown to modulate the expressionof HIF-1α and pharmaceutical preparations thereof and their use astreatment of cancer diseases and pre-eclampsia are disclosed.

BACKGROUND OF THE INVENTION

Solid tumors must establish a blood supply and have enhanced glucosemetabolism to grow beyond a few millimeters. How they sense hypoxia, andrespond by activating hypoxia-inducible genes and secreting angiogenicfactors to establish a blood system is central to cancer biology. Manytumors contain hypoxic microenvironments, which have been associatedwith malignant progression, metastasis and resistance to radiotherapyand chemotherapy.

The discovery of hypoxia-inducible factor-1 (HIF-1) gave some insightinto the regulation of hypoxia-inducible genes (U.S. Pat. No. 5,882,914and WO9639426; WO9948916). HIF-1 is composed of two subunits HIF-1α andHIF-10 and it binds hypoxia-response elements (HREs) in enhancers ofgenes encoding angiogenic factors such as VEGF and glycolysis-relatedproteins such as glycolytic enzymes and glucose transporter 1 and 3(GLU-1 and 3).

It has been demonstrated that engineered down-regulation of HIF-1α byintratumoral gene transfer of an antisense HIF-1α plasmid leads to thedown-regulation of VEGF, and decreased tumor microvessel density (WO0076497, Sun X et al, Gene Therapy (2001) 8, 638-645). The plasmidcontained a 320-bp cDNA fragment encoding 5′-end of HIF-1α (nucleotides152-454; Genebank AF003698). Furthermore, in the International PatentApplication cited above a method was described based on that theexpression vector should be used in conjunction with animmunotherapeutic agent. However, a major weakness with the expressionplasmid approach is that it will not be suitable as a therapeutic agentdue to its size and the nuclease sensitivity of the expression product.

Besides the plasmid expressing a HIF-1α fragment a few antisenseoligonucleotides targeting HIF-1α have been designed as research toolsto study a specific biological mechanism or biological target. Forexample the antisense inhibition of HIF-1α expression in hypoxicexplants have been shown to inhibit expression of TGFβ (Caniggia, I., etal J. of Clinical Investigation, March 2000, 105, 577-587). In thisparticular study, only one antisense oligonucleotide was synthesized, aphosphorothioate targeted against the sequence adjacent to the AUGinitiation codon of HIF-1α mRNA. The sequences were HIF-1α5′-GCCGGCGCCCTCCAT-3′ and the HIF-1α down regulation was demonstrated atmRNA level. This oligo has been used to study the role of HIF-1α inextravillous trophoblast outgrowth and invasion, and implicated atpotential role of HIF-1α in pre-eclampsia (Caniggia, I. et al Placenta(2000), 21, Supplement A, Trophoblast Research 14, S25-S30).

Another study, using the same oligonucleotide sequence as above, showedthat antisense inhibition of HIF-1α resulted in loss of peroxisomeproliferator-active receptors (PPARs) (Narravula, S. and Colgan S P., J.of Immunology, 2001, 166, 7543-7548). The above mentioned oligo has alsobeen used to show that nickel requires HIF-1α to induce plasminogenactivator inhibitior-1 (PAI-1) (Andrew, A. S. Klei L. R., Barchowsky A,Am. J. Physiol. Lung Cell Mol. Physiol. 281, L607-L615, 2001).

A single antisense oligonucleotide has also been used to study the twosplice variants of the hypoxia-inducible factor HIF-1 cc as potentialdimerization partner of ARNT2 in neurons. The antisense oligonucleotidewas the phosphorothioate-modification of the sequence:5′-TCTTCTCGTTCTCGCC-3′. Treating cells with this oligonucleotideresulted in inhibition of [³H]thymidine incorporation, but did not havean effect on apoptosis in normoxic cells (Drutel et. al. (2000) Eur. J.Neurosci. 12, 3701-3708).

Furthermore, a single antisense oligonucleotide for HIF-1α have beenshowed to inhibit the increased gene expression of cardiac endothelin(ET)-1 and it was hypothesized that HIF-1α is involved in increasedmyocardial expression of the ET-1 gene in heart failure (Kakinuma, Y. etal, Circulation, 2001; 103, 2387-2394). The anti sense oligonucleotidehad the following sequence:

CCTCCATGGCGAATCGGTGC.

Currently, there are no known therapeutic antisense agents, whicheffectively inhibit the synthesis of HIF-1α and which can be used forthe treatment of a disease. Consequently, there is a need for agentscapable of effectively inhibiting the HIF-1α function to be used in thetreatment of e.g. cancer and pre-eclampsia.

SUMMARY OF THE INVENTION

The present invention is directed to oligomeric compounds, particularlyLNA antisense oligonucleotides, which are targeted to a nucleic acidencoding HIF-1α and which modulate the expression of the HIF-1α.Pharmaceutical and other compositions comprising the oligomericcompounds of the invention are also provided. Further provided aremethods of modulating the expression of HIF-1α in cells or tissuescomprising contacting said cells or tissues with one or more of theoligomeric compounds or compositions of the invention. Also disclosedare methods of treating an animal or a human, suspected of having orbeing prone to a disease or condition, associated with expression ofHIF-1α by administering a therapeutically or prophylactically effectiveamount of one or more of the oligomeric compounds or compositions of theinvention. Further, methods of using oligomeric compounds for theinhibition of expression of HIF-1α and for treatment of diseasesassociated with these HIF-1α are provided. Examples of such diseases aredifferent types of cancer, particularly common cancers, as e.g. primaryand metastatic breast, colorectal, prostate, pancreas, other GI-cancers,lung, cervical, ovarian, and brain tumors, as well as pre-eclampsia,inflammatory bowel disease and Alzheimers disease. Other examples arecancer of the colon, liver, thyroid, kidney, testes, stomach, intestine,bowel, esophagus, spinal cord, sinuses, bladder or urinary tract.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a Western blot of HIF-1α protein. Cells were treated withthe different oligos at 100 nM for 4 hours. The cells were allowed togrow for 18 hours before they were exposed to severe hypoxia for 6hours.

FIG. 2 shows a Western blot of HIF-1α protein. U87 cells were treatedwith three of the oligos at 200 nM for 4 hours. The cells were exposedto severe hypoxia for 18 hours immediately after the treatment.

FIG. 3 shows Western blots of HIF-1α, VEGF Glut 1 and tubulin protein inU87 cells treated with oligo Cur0813. Cells were treated with oligo for24 hours at 100 nM, 200 nM, 300 nM and 400 nM. The cells were exposed tosevere hypoxia for 18 hours immediately after the treatment.

FIG. 4 shows Western blots of HIF-1α and tubulin protein in U87 cellstreated with mismatch oligos (Cur0960 and Cur0961). Cells were treatedwith oligo for 24 hours at 100 nM, 200 nM, 300 nM and 400 nM. The cellswere exposed to severe hypoxia for 18 hours immediately after thetreatment.

FIG. 5 shows Western blots of HIF-1α, VEGF and tubulin protein in 15PC3cells treated with oligo Cur813. Cells were treated with oligo for 16hours at 125 nM, 25 nM, 5 nM and 1 nM. The cells were exposed to severehypoxia for 6 hours immediately after the treatment.

FIG. 6 shows Western blots of HIF-1α and tubulin protein in 15PC3 cellstreated with different oligos at 5 nM for 16 hours. The cells wereexposed to severe hypoxia for 6 hours immediately after the treatment.

FIG. 7 shows Western blots of HIF-1α and tubulin protein in U373 cellstreated with different oligos at 100 nM for 6 hours. The cells wereexposed to severe hypoxia for 20 hours immediately after the treatment.

FIG. 8 shows Western blots of HIF-1α and tubulin protein in U373 cellstreated with different oligos at 100 nM for 6 hours. The cells wereexposed to severe hypoxia for 20 hours immediately after the treatment.

FIG. 9 shows growth curves of U373 xenograft tumours treated with PBS orCur813 at 5 mg/kg/day i.p. 1× daily for 7 days. Bars represent standarderrors.

FIG. 10 shows human. HIF-1α sequence, using GenBank accession numberNM_(—)001530, incorporated herein as SEQ ID NO:1.

DEFINITION

As used herein, the terms “target nucleic acid” encompass DNA encodingthe hypoxia-inducible factor or encoding hypoxia-inducible factor-1α(HIF-1α), RNA (including pre-mRNA and mRNA) transcribed from such DNA,and also cDNA derived from such RNA.

As used herein, the term “gene” means the gene including exons, introns,non-coding 5′ and 3′ regions and regulatory elements and all currentlyknown variants thereof and any further variants, which may beelucidated.

As used herein, the terms “oligomeric compound” refers to anoligonucleotide which can induce a desired therapeutic effect in humansthrough for example binding by hydrogen bonding to either a target gene“Chimeraplast” and “TFO”, to the RNA transcript(s) of the target gene“antisense inhibitors”, “siRNA”, “ribozymes” and oligozymes” or to theprotein(s) encoding by the target gene “aptamer”, spiegelmer” or“decoy”.

As used herein, the term “mRNA” means the presently known mRNAtranscript(s) of a targeted gene, and any further transcripts, which maybe identified.

As used herein, the term “modulation” means either an increase(stimulation) or a decrease (inhibition) in the expression of a gene. Inthe present invention, inhibition is the preferred form of modulation ofgene expression and mRNA is a preferred target.

As used herein, the term “targeting” an antisense compound to aparticular target nucleic acid means providing the antisenseoligonucleotide to the cell, animal or human in such a way that theantisense compound are able to bind to and modulate the function of itsintended target.

As used herein, “hybridisation” means hydrogen bonding, which may beWatson-Crick, Hoogsteen, reversed Hoogsteen hydrogen bonding, etc.between complementary nucleoside or nucleotide bases. Watson and Crickshowed approximately fifty years ago that deoxyribo nucleic acid (DNA)is composed of two strands which are held together in a helicalconfiguration by hydrogen bonds formed between opposing complementarynucleobases in the two strands. The four nucleobases, commonly found inDNA are guanine (G), adenine (A), thymine (T) and cytosine (C) of whichthe G nucleobase pairs with C, and the A nucleobase pairs with T. In RNAthe nucleobase thymine is replaced by the nucleobase uracil (U), whichsimilarly to the T nucleobase pairs with A. The chemical groups in thenucleobases that participate in standard duplex formation constitute theWatson-Crick face. Hoogsteen showed a couple of years later that thepurine nucleobases (G and A) in addition to their Watson-Crick face havea Hoogsteen face that can be recognised from the outside of a duplex,and used to bind pyrimidine oligonucleotides via hydrogen bonding,thereby forming a triple helix structure.

In the context of the present invention “complementary” refers to thecapacity for precise pairing between two nucleotides or nucleosidesequences with one another. For example, if a nucleotide at a certainposition of an oligonucleotide is capable of hydrogen bonding with anucleotide at the corresponding position of a DNA or RNA molecule, thenthe oligonucleotide and the DNA or RNA are considered to becomplementary to each other at that position. The DNA or RNA and theoligonucleotide are considered complementary to each other when asufficient number of nucleotides in the oligonucleotide can formhydrogen bonds with corresponding nucleotides in the target DNA or RNAto enable the formation of a stable complex. To be stable in vitro or invivo the sequence of an antisense compound need not be 100%complementary to its target nucleic acid. The terms “complementary” and“specifically hybridisable” thus imply that the antisense compound bindssufficiently strongly and specifically to the target molecule to providethe desired interference with the normal function of the target whilstleaving the function of non-target mRNAs unaffected.

The term “Nucleic Acid Analogues” refers to a non-natural nucleic acidbinding compound. Nucleic Acid Analogues are described in e.g. Freier &Altmann (Nucl. Acid Res., 1997, 25, 4429-4443) and Uhlmann (Curr.Opinion in Drug & Development (2000, 3(2): 293-213). Scheme 1illustrates selected examples.

The term “LNA” refers to an oligonucleotide containing one or morebicyclic nucleoside analogues also referred to as a LNA monomer. LNAmonomers are described in WO 9914226 and subsequent applications,WO0056746, WO0056748, WO0066604, WO00125248, WO0228875, WO2002094250 andPCT/DK02/00488. One particular example of a thymidine LNA monomer is the(1S,3R,4R,7S)-7-hydroxy-1-hydroxymethyl-5-methyl-3-(thymin-1yl)-2,5-dioxa-bicyclo[2:2:1]heptane.

The term “oligonucleotide” refers, in the context of the presentinvention, to an oligomer (also called oligo) or nucleic acid polymer(e.g. ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) or nucleicacid analogue of those known in the art, preferably Locked Nucleic Acid(LNA), or a mixture thereof. This term includes oligonucleotidescomposed of naturally occurring nucleobases, sugars and internucleoside(backbone) linkages as well as oligonucleotides havingnon-naturally-occurring portions which function similarly or withspecific improved functions. A fully or partly modified or substitutedoligonucleotides are often preferred over native forms because ofseveral desirable properties of such oligonucleotides such as forinstance, the ability to penetrate a cell membrane, good resistance toextra- and intracellular nucleases, high affinity and specificity forthe nucleic acid target. The LNA analogue is particularly preferredexhibiting the above-mentioned properties.

By the term “unit” is understood a monomer.

The term “at least one” comprises the integers larger than or equal to1, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 andso forth.

The term “thio-LNA” comprises a locked nucleotide in which at least oneof X or Y in Scheme 2 is selected from S or —CH₂—S—. Thio-LNA can be inboth beta-D and alpha-L-configuration.

The term “amino-LNA” comprises a locked nucleotide in which at least oneof X or Y in Scheme 2-N(H)—, CH₂—N(H)—, —CH₂—N(R)— where R is selectedfrom hydrogen and C₁₋₄-alkyl. Amino-LNA can be in both beta-D andalpha-L-configuration.

The term “oxy-LNA” comprises a locked nucleotide in which at least oneof X or Y in Scheme 2 represents O or —CH₂—O—. Oxy-LNA can be in bothbeta-D and alpha-L-configuration.

The term “ena-LNA” comprises a locked nucleotide in which Y in Scheme 2is —CH₂—O—.

By the term “alpha-L-LNA” comprises a locked nucleotide represented asshown in Scheme 3.

By the term “LNA derivatives” comprises all locked nucleotide in Scheme2 except beta-D-methylene LNA e.g. thio-LNA, amino-LNA, alpha-L-oxy-LNAand ena-LNA.

DETAILED DESCRIPTION OF THE INVENTION

The present invention employs oligomeric compounds, particularlyantisense oligonucleotides, for use in modulating the function ofnucleic acid molecules encoding HIF-1α. The modulation is ultimately achange in the amount of HIF-1α produced. In one embodiment this isaccomplished by providing antisense compounds, which specificallyhybridise with nucleic acids encoding HIF-1α. The modulation ispreferably an inhibition of the expression of HIF-1α, which leads to adecrease in the number of functional proteins produced. HIF-1 may beinvolved in angiogenesis as well as red blood cell proliferation,cellular proliferation, iron metabolism, glucose and energy metabolism,pH regulation, tissue invasion, apoptosis, multi-drug resistance,cellular stress response or matrix metabolism.

Antisense and other oligomeric compounds of the invention, whichmodulate expression of the target, are identified throughexperimentation or though rational design based on sequence informationon the target and know-how on how best to design an oligomeric compoundagainst a desired target. The sequences of these compounds are preferredembodiments of the invention. Likewise, the sequence motifs in thetarget to which these preferred oligomeric compounds are complementary(referred to as “hot spots”) are preferred sites for targeting.

Preferred oligomeric compounds according to the invention are SEQ ID NO2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114 or 115 and their sequences are presented intable 1 and table 2. The oligomeric compounds according to the inventionare potent modulators of target. This is showed experimentally both invitro and in vivo. In vitro inhibition of target is shown in table 1 andFIG. 1-8 using three different cancer cell lines. FIG. 9 shows in vivadown regulation of target. Furthermore, the oligomeric compounds areshown to be potent inhibitors in much lower concentration than e.g. thestandard condition for phosphorthioate antisense oligonucleotides. FIGS.5 and 6 show inhibition of compounds of the invention down to 5 nM.Inhibition of HIF-1α by oligomeric compounds of the invention can alsoinhibit the expression of Vascular Endothelial Growth Factor (VEGF)known to be involved in angiogenesis and Glucose Transporter-1 (GLUT-1)known to be involved in glucose uptake as shown in FIGS. 3 and 5.Various designs of oligomeric compounds shown in table 2 targeted to twomotifs were identified as potent inhibitors of the target as shown inFIGS. 1 and 7. A genewalk was performed using oligomeric compounds fromtable 1, and the effect of the potent oligomeric compounds is shown inFIG. 8. All the above-mentioned experimental observations show that thecompounds according to the invention can constitute the active compoundin a pharmaceutical composition.

Furthermore, the oligomeric compounds according to the invention mayinhibit HIF-1α under normoxia and hypoxia.

In one embodiment of the invention the oligomeric compounds arecontaining at least on unit of chemistry termed LNA (Locked NucleicAcid).

LNA monomer typically refers to a bicyclic nucleoside analogue, asdescribed in the International Patent Application WO 99/14226 andsubsequent applications, WO0056746, WO0056748, WO0066604, WO00125248,WO0228875, WO2002094250 and PCT/DK02/00488 all incorporated herein byreference. Preferred LNA monomers structures are exemplified in Scheme 2

X and Y are independently selected among the groups —O—, —S—, —N(H)—,N(R)—, —CH₂— or —CH— (if part of a double bond), —CH₂—O—, —CH₂—S—,—CH₂—N(R)—, —CH₂—CH₂— or —CH₂—CH— (if part of a double bond), —CH═CH—,where R is selected from hydrogen and C₁₋₄-alkyl. The asymmetric groupsmay be found in either orientation.

In Scheme 2 the 4 chiral centers are shown in a fixed configuration.However, also comprised in this invention are compounds of the generalScheme 2 in which the chiral centers are found in differentconfigurations. Thus, each chiral center in Scheme 2 can exist in eitherR or S configuration. The definition of R (rectus) and S (sinister) aredescribed in the IUPAC 1974 Recommendations, Section E, FundamentalStereochemistry The rules can be found in Pure Appl. Chem. 45, 13-30,(1976) and in “Nomenclature of organic Chemistry” Pergamon, New York,1979. Z and Z* are independently selected among an internucleosidelinkage, a terminal group or a protecting group.

The internucleoside linkage may be —O—P(O)₂—O—, —O—P(O,S)—O—,—O—P(S)₂—O—, —S—P(O)₂—O—, —S—P(O,S)—O—, —S—P(S)₂—O—, —O—P(O)₂—S—,—O—P(O,S)—S—, —S—P(O)₂—S—, —O—PO(R^(H))—O—, O—PO(OCH₃)—O—,—O—PO(NR^(H))—O—, —O—PO(OCH₂CH₂S—R)—O—, —O—PO(BH₃)—O—,—O—PO(NHR^(H))—O—, —O—P(O)₂—NR^(H)—, —NR^(H)—P(O)₂—O—, —NR^(H)—CO—O—,—NR^(H)—CO—NR^(H)—, —O—CO—O—, —O—CO—NR^(H)—, —O—CH₂—CO—NR^(H)—,—O—CH₂—CH₂—NR^(H)—, —CO—NR^(H)—CH₂—, —CH₂—NR^(H)—CO—, —O—CH₂—CH₂—S—,—S—CH₂—CH₂—O—, —S—CH₂—CH₂—S—, —CH₂—SO₂—CH₂—, —CH₂—CO—NR^(H)—,—O—CH₂—CH₂—NR^(H)—CO —CH₂—NCH₃—O—CH₂—, where R^(H) is selected formhydrogen and C₁₋₄-alkyl,

The terminal groups are selected independently from hydrogen, azido,halogen, cyano, nitro, hydroxy, Prot-O—, Act-O—, mercapto, Prot-S—,Act-S—, C₁₋₆-alkylthio, amino, Prot-N(R^(H))—, Act-N(R^(H))—, mono- ordi(C₁₋₆-alkyl)amino, optionally substituted C₁₋₆-alkoxy, optionallysubstituted C₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl, optionallysubstituted C₂₋₆-alkenyloxy, optionally substituted C₂₋₆-alkynyl,optionally substituted C₂₋₆-alkynyloxy, monophosphate-or protectedmonophosphate, monothiophosphate-or protected monothiophosphate,diphosphate-or protected diphosphate, dithiophosphate-or protecteddithiophosphate, triphosphate- or protected triphosphate,trithiophosphate-or protected trithiophosphate. Examples of suchprotection groups on the phosphate residues are S-acetylthioethyl (SATE)or 5-pivaloylthioethyl (t-butyl-SATE), DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, ligands, carboxy, sulphono, hydroxymethyl,Prot-O—CH₂—, Act-O—CH₂—, aminomethyl,Prot-N(R^(H))—CH₂-Act-N(R^(H))—CH₂—, carboxymethyl, sulphonomethyl,where Prot is a protection group for —OH, —SH, and —NH(R^(H)),respectively, Act is an activation group for —OH, —SH, and —NH(R^(H)),respectively, and R^(H) is selected from hydrogen and C₁₋₄-alkyl;

The protection groups of hydroxy substituents comprises substitutedtrityl, such as 4,4′-dimethoxytrityloxy (DMT), 4-monomethoxytrityloxy(MMT), and trityloxy, optionally substituted 9-(9-phenyl)xanthenyloxy(pixyl), optionally substituted methoxytetrahydropyranyloxy (mthp),silyloxy such as trimethylsilyloxy (TMS), triisopropylsilyloxy (TIPS),tert-butyldimethylsilyloxy (TBDMS), triethylsilyloxy, andphenyldimethylsilyloxy, tert-butylethers, acetals (including two hydroxygroups), acyloxy such as acetyl or halogen substituted acetyls, e.g.chloroacetyloxy or fluoroacetyloxy, isobutyryloxy, pivaloyloxy,benzoyloxy and substituted benzoyls, methoxymethyloxy (MOM), benzylethers or substituted benzyl ethers such as 2,6-dichlorobenzyloxy(2,6-O₂Bzl). Alternatively when Z or Z* is hydroxyl they may beprotected by attachment to a solid support optionally through a linker.

When Z or Z* is amino groups illustrative examples of the aminoprotection protections are fluorenylmethoxycarbonylamino (Fmoc),tert-butyloxycarbonylamino (BOC), trifluoroacetylamino,allyloxycarbonylamino (alloc, AOC), Z benzyl-oxycarbonylamino (Cbz),substituted benzyloxycarbonylaminos such as 2-chlorobenzyloxycarbonylamino (2-ClZ), monomethoxytritylamino (MMT),dimethoxytritylamino (DMT), phthaloylamino, and9-(9-phenyl)xanthenylamino (pixyl).

In the embodiment above, Act designates an activation group for —OH,—SH, and —NH(R^(H)). In a preferred embodiment such activators mediatescouplings to other residues, monomers. After such successful couplingsthe act-group is converted to an internucleoside linkage. Suchactivation groups are, e.g., selected from optionally substitutedO-phosphoramidite, optionally substituted O-phosphortriester, optionallysubstituted O-phosphordiester, optionally substituted H-phosphonate, andoptionally substituted O-phosphonate.

In the present context, the term “phosphoramidite” means a group of theformula —P(OR^(x))—N(R^(y))₂, wherein R^(x) designates an optionallysubstituted alkyl group, e.g. methyl, 2-cyanoethyl, or benzyl, and eachof R^(y) designate optionally substituted alkyl groups, e.g. ethyl orisopropyl, or the group —N(R^(y))₂ forms a morpholino group(—N(CH₂CH₂)₂O). R^(x) preferably designates 2-cyanoethyl and the twoR^(y) are preferably identical and designate isopropyl. Thus, anespecially relevant phosphoramidite isN,N-diisopropyl-O-(2-cyanoethyl)phosphoramidite.

B constitutes a natural or non-natural nucleobase and selected amongadenine, cytosine, 5-methylcytosine, isocytosine, pseudoisocytosine,guanine, thymine, uracil, 5-bromouracil, 5-propynyluracil,5-propyny-6-fluoroluracil, 5-methylthiazoleuracil, 6-aminopurine,2-aminopurine, inosine, diaminopurine, 7-propyne-7-deazaadenine,7-propyne-7-deazaguanine, 2-chloro-6-aminopurine.

Particularly preferred bicyclic structures are shown in Scheme 3 below:

Where X is —O—, —S—, —NH—, and N(R^(H)),

Z and Z* are independently selected among an internucleoside linkage, aterminal group or a protecting group.

The internucleotide linkage may be —O—P(O)₂—O—, —O—P(O,S)—O—,—O—P(S)₂—O—, —S—P(O)₂—O—, —S—P(O,S)—O—, —S—P(S)₂—O—, —O—P(O)₂—S—,—O—P(O,S)—S—, —S—P(O)₂—S—, —O—PO(R^(H))—O—, O—PO(OCH₃)—O—,—O—PO(NR^(H))—O—, —O—PO(OCH₂CH₂S—R)—O—, —O—PO(BH₃)—O—,—O—PO(NHR^(H))—O—, —O—P(O)₂—NR^(H)—, —NR^(H)—P(O)₂—O—, —NR^(H)—CO—O—,where R^(H) is selected form hydrogen and C₁₋₄-alkyl.

The terminal groups are selected independently among from hydrogen,azido, halogen, cyano, nitro, hydroxy, Prot-O—, Act-O—, mercapto,Prot-S—, Act-S—, C₁₋₆-alkylthio, amino, Prot-N(R^(H)—, Act-N(R) ^(H))—,mono- or di(C₁₋₆-alkyl)amino, optionally substituted C₁₋₆-alkoxy,optionally substituted C₁₋₄-alkyl, optionally substituted monophosphate,monothiophosphate, diphosphate, dithiophosphate triphosphate,trithiophosphate, where Prot is a protection group for —OH, —SH, and—NH(R^(H)), respectively, Act is an activation group for —OH, —SH, and—NH(O), respectively, and R^(H) is selected from hydrogen andC₁₋₆-alkyl.

The protection groups of hydroxy substituents comprises substitutedtrityl, such as 4,4′-dimethoxytrityloxy (DMT), 4-monomethoxytrityloxy(MMT), optionally substituted 9-(9-phenyl)xanthenyloxy (pixyl),optionally substituted methoxytetrahydropyranyloxy (mthp), silyloxy suchas trimethylsilyloxy (TMS), triisopropylsilyloxy (TIPS),tert-butyldimethylsilyloxy (TBDMS), triethylsilyloxy, andphenyldimethylsilyloxy, tert-butylethers, acetals (including two hydroxygroups), acyloxy such as acetyl Alternatively when Z or Z* is hydroxylthey may be protected by attachment to a solid support optionallythrough a linker.

When Z or Z* is amino groups illustrative examples of the aminoprotection protections are fluorenylmethoxycarbonylamino (Fmoc),tert-butyloxycarbonylamino (BOC), trifluoroacetylamino,allyloxycarbonylamino (alloc, AOC), monomethoxytritylamino (MMT),dimethoxytritylamino (DMT), phthaloylamino.

In the embodiment above, Act designates an activation group for —OH,—SH, and —NH(R^(H)). In a preferred embodiment such activators mediatescouplings to other residues, monomers. After such successful couplingsthe act-group is converted to an internucleoside linkage. Suchactivation groups are, e.g., selected from optionally substitutedO-phosphoramidite, optionally substituted O-phosphortriester, optionallysubstituted O-phosphordiester, optionally substituted H-phosphonate, andoptionally substituted O-phosphonate.

In the present context, the term “phosphoramidite” means a group of theformula —P(OR^(x))—N(R^(y))₂, wherein R^(x) designates an optionallysubstituted alkyl group, e.g. methyl, 2-cyanoethyl, and each of R^(y)designate optionally substituted alkyl groups, R^(x) preferablydesignates 2-cyanoethyl and the two R^(y) are preferably identical anddesignate isopropyl. Thus, an especially relevant phosphoramidite isN,N-diisopropyl-O-(2-cyanoethyl)phosphoramidite.

B constitutes a natural or non-natural nucleobase and selected amongadenine, cytosine, 5-methylcytosine, isocytosine, pseudoisocytosine,guanine, thymine, uracil, 5-bromouracil, 5-propynyluracil,6-aminopurine, 2-aminopurine, inosine, diaminopurine,2-chloro-6-aminopurine.

Therapeutic Principle

A person skilled in the art will appreciate that oligomeric compoundscontaining LNA can be used to combat HIF-1α linked diseases by manydifferent principles, which thus falls within the spirit of the presentinvention.

For instance, LNA oligomeric compounds may be designed as antisenseinhibitors, which are single stranded nucleic acids that prevent theproduction of a disease causing protein, by intervention at the mRNAlevel. Also, they may be designed as Ribozymes or Oligozymes which areantisense oligonucleotides which in addition to the target bindingdomain(s) comprise a catalytic activity that degrades the target mRNA(ribozymes) or comprise an external guide sequence (EGS) that recruit anendogenous enzyme (RNase P) which degrades the target mRNA (oligozymes)

Equally well, the LNA oligomeric compounds may be designed as siRNA'swhich are small double stranded RNA molecules that are used by cells tosilence specific endogenous or exogenous genes by an as yet poorlyunderstood “antisense-like” mechanism.

LNA oligomeric compounds may also be designed as Aptamers (and avariation thereof, termed Spiegelmers) which are nucleic acids thatthrough intra-molecular hydrogen bonding adopt three-dimensionalstructures that enable them to bind to and block their biologicaltargets with high affinity and specificity. Also, LNA oligomericcompounds may be designed as Decoys, which are small double-strandednucleic acids that prevent cellular transcription factors fromtransactivating their target genes by selectively blocking their DNAbinding site.

Furthermore, LNA oligomeric compounds may be designed as Chimeraplasts,which are small single stranded nucleic acids that are able tospecifically pair with and alter a target gene sequence. LNA containingoligomeric compounds exploiting this principle therefore may beparticularly useful for treating HIF-1α linked diseases that are causedby a mutation in the HIF-1α gene.

Finally, LNA oligomeric compounds may be designed as TFO's (triplexforming oligonucleotides), which are nucleic acids that bind to doublestranded DNA and prevent the production of a disease causing protein, byintervention at the RNA transcription level.

Dictated in part by the therapeutic principle by which theoligonucleotide is intended to operate, the LNA oligomeric compounds inaccordance with this invention preferably comprise from about 8 to about60 nucleobases i.e. from about 8 to about 60 linked nucleosides.Particularly preferred compounds are antisense oligonucleotidescomprising from about 12 to about 30 nucleobases and most preferably areantisense compounds comprising about 12-20 nucleobases.

Referring to the above principles by which an LNA oligomeric compoundcan elicit its therapeutic action the target of the present inventionmay be the HIF-1α gene, the mRNA or the protein. In the most preferredembodiment the LNA oligomeric compounds is designed as an antisenseinhibitor directed against the HIF-1α pre-mRNA or HIF-1α mRNA. Theoligonucleotides may hybridize to any site along the HIF-1α pre-mRNA ormRNA such as sites in the 5′ untranslated leader, exons, introns and 3′untranslated tail.

In a preferred embodiment, the oligonucleotide hybridizes to a portionof the human HIF-1α pre-mRNA or mRNA that comprises thetranslation-initiation site. More preferably, the HIF-1α oligonucleotidecomprises a CAT sequence, which is complementary to the AUG initiationsequence of the HIF-1α pre-mRNA or RNA. In another embodiment, theHIF-1α oligonucleotide hybridizes to a portion of the splice donor siteof the human HIF-1α pre-mRNA. In yet another embodiment,HIF-1αoligonucleotide hybridizes to a portion of the splice acceptorsite of the human HIF-1α pre-mRNA. In another embodiment, the HIF-1αoligonucleotide hybridizes to portions of the human HIF-1α pre-mRNA ormRNA involved in polyadenylation, transport or degradation.

The skilled person will appreciate that preferred oligonucleotides arethose that hybridize to a portion of the HIF-1α pre-mRNA or mRNA whosesequence does not commonly occur in transcripts from unrelated genes soas to maintain treatment specificity.

The oligomeric compound of the invention are designed to be sufficientlycomplementary to the target to provide the desired clinical responsee.g. the oligomeric compound must bind with sufficient strength andspecificity to its target to give the desired effect. In one embodiment,said compound modulating HIF-1α is designed so as to also modulate otherspecific nucleic acids which do not encode HIF-1α.

It is preferred that the oligomeric compound according to the inventionis designed so that intra- and intermolecular oligonucleotidehybridisation is avoided.

In many cases the identification of an LNA oligomeric compound effectivein modulating HIF-1α activity in vivo or clinically is based on sequenceinformation on the target gene. However, one of ordinary skill in theart will appreciate that such oligomeric compounds can also beidentified by empirical testing. As such HIF-1α oligomeric compoundshaving, for example, less sequence homology, greater or fewer modifiednucleotides, or longer or shorter lengths, compared to those of thepreferred embodiments, but which nevertheless demonstrate responses inclinical treatments, are also within the scope of the invention.

Antisense Drugs

In one embodiment of the invention the oligomeric compounds are suitableantisense drugs. The design of a potent and safe antisense drug requiresthe fine-tuning of diverse parameters such as affinity/specificity,stability in biological fluids, cellular uptake, mode of action,pharmacokinetic properties and toxicity.

Affinity & Specificity:

LNA with an oxymethylene 4′-C linkage (β-D-oxy-LNA), exhibitsunprecedented binding properties towards DNA and RNA target sequences.Likewise LNA derivatives, such as amino-, thio- and α-L-oxy-LNA displayunprecedented affinities towards complementary RNA and DNA and in thecase of thio-LNA the affinity towards RNA is even better than with theβ-D-oxy-LNA.

In addition to these remarkable hybridization properties, LNA monomerscan be mixed and act cooperatively with DNA and RNA monomers, and withother nucleic acid analogues, such as 2′-O-alkyl modified RNA monomers.As such, the oligonucleotides of the present invention can be composedentirely of β-D-oxy-LNA monomers or it may be composed of β-D-oxy-LNA inany combination with DNA, RNA or contemporary nucleic acid analogueswhich includes LNA derivatives such as for instance amino-, thio- andα-L-oxy-LNA. The unprecedented binding affinity of LNA towards DNA orRNA target sequences and its ability to mix freely with DNA, RNA and arange of contemporary nucleic acid analogues has a range of importantconsequences according to the invention for the development of effectiveand safe antisense compounds.

Firstly, in one embodiment of the invention it enables a considerableshortening of the usual length of an antisense oligo (from 20-25 mersto, e.g., 12-15 mers) without compromising the affinity required forpharmacological activity. As the intrinsic specificity of an oligo isinversely correlated to its length, such a shortening will significantlyincrease the specificity of the antisense compound towards its RNAtarget. One embodiment of the invention is to, due to the sequence ofthe human genome is available and the annotation of its genes rapidlyprogressing, identify the shortest possible, unique sequences in thetarget mRNA.

In another embodiment, the use of LNA to reduce the size of oligossignificantly eases the process and prize of manufacture thus providingthe basis for antisense therapy to become a commercially competitivetreatment offer for a diversity of diseases.

In another embodiment, the unprecedented affinity of LNA can be used tosubstantially enhance the ability of an antisense oligo to hybridize toits target mRNA in-vivo thus significantly reducing the time and effortrequired for identifying an active compound as compared to the situationwith other chemistries.

In another embodiment, the unprecedented affinity of LNA is used toenhance the potency of antisense oligonucleotides thus enabling thedevelopment of compounds with more favorable therapeutic windows thanthose currently in clinical trials.

When designed as an antisense inhibitor, the oligonucleotides of theinvention bind to the target nucleic acid and modulate the expression ofits cognate protein. Preferably, such modulation produces an inhibitionof expression of at least 10% or 20% compared to the normal expressionlevel, more preferably at least a 30%, 40%, 50%, 60%, 70%, 80%, or 90%inhibition compared to the normal expression level.

Typically, the LNA oligonucleotides of the invention will contain otherresidues than β-D-oxy-LNA such as native DNA monomers, RNA monomers,N3′-P5′phosphoroamidates, 2′-F, 2′-O-Me, 2′-O-methoxyethyl (MOE),2′-O-(3-aminopropyl) (AP), hexitol nucleic acid (HNA), V-F-arabinonucleic acid (2′-F-ANA) and D-cyclohexenyl nucleoside (CeNA). Also, theβ-D-oxy-LNA-modified oligonucleotide may also contain other LNA units inaddition to or in place of an oxy-LNA group. In particular, preferredadditional LNA units include thio-LNA or amino-LNA monomers in eitherthe D-β or L-α configurations or combinations thereof or ena-LNA. Ingeneral, an LNA-modified oligonucleotide will contain at least about 5,10, 15 or 20 percent LNA units, based on total nucleotides of theoligonucleotide, more typically at least about 20, 25, 30, 40, 50, 60,70, 80 or 90 percent LNA units, based on total bases of theoligonucleotide.

Stability in Biological Fluids:

One embodiment of the invention includes the incorporation of LNAmonomers into a standard DNA or RNA oligonucleotide to increase thestability of the resulting oligomeric compound in biological fluids e.g.through the increase of resistance towards nucleases (endonucleases andexonucleases). The extent of stability will depend on the number of LNAmonomers used, their position in the oligonucleotide and the type of LNAmonomer used. Compared to DNA and phosphorothioates the following orderof ability to stabilize an oligonucleotide against nucleolyticdegradation can be established: DNA<<phosphorothioates˜oxy-LNA<α-L-LNA<amino-LNA <thio-LNA.

Given the fact that LNA is compatible with standard DNA synthesis andmixes freely with many contemporary nucleic acid analogues nucleaseresistance of LNA- oligomeric compounds can be further enhancedaccording to the invention by either incorporating other analogues thatdisplay increased nuclease stability or by exploiting nuclease-resistantinternucleoside linkages e.g. phosphoromonothioate, phosphorodithioate,and methylphosphonate linkages, etc.

Mode of Action:

Antisense compounds according to the invention may elicit theirtherapeutic action via a variety of mechanisms and may be able tocombine several of these in the same compound. In one scenario, bindingof the oligonucleotide to its target (pre-mRNA or mRNA) acts to preventbinding of other factors (proteins, other nucleic acids, etc.) neededfor the proper function of the target i.e. operate by steric hindrance.For instance, the antisense oligonucleotide may bind to sequence motifsin either the pre-mRNA or mRNA that are important for recognition andbinding of transacting factors involved in splicing, poly-adenylation,cellular transport, post-transcriptional modifications of nucleosides inthe RNA, capping of the 5′-end, translation, etc. In the case ofpre-mRNA splicing, the outcome of the interaction between theoligonucleotide and its target may be either suppression of expressionof an undesired protein, generation of alternative spliced mRNA encodinga desired protein or both. In another embodiment, binding of theoligonucleotide to its target disables the translation process bycreating a physical block to the ribosomal machinery, i.e. tranlationalarrest.

In yet another embodiment, binding of the oligonucleotide to its targetinterferes with the RNAs ability to adopt secondary and higher orderstructures that are important for its proper function, i.e. structuralinterference. For instance, the oligonucleotide may interfere with theformation of stem-loop structures that play crucial roles in differentfunctions, such as providing additional stability to the RNA or adoptingessential recognition motifs for different proteins.

In still another embodiment, binding of the oligonucleotide inactivatesthe target toward further cellular metabolic processes by recruitingcellular enzymes that degrades the mRNA. For instance, theoligonucleotide may comprise a segment of nucleosides that have theability to recruit ribonuclease H(RNaseH) that degrades the RNA part ofa DNA/RNA duplex. Likewise, the oligonucleotide may comprise a segmentwhich recruits double stranded RNAses, such as for instance RNAseIII orit may comprise an external guide sequence (EGS) that recruit anendogenous enzyme (RNase P) which degrades the target mRNA. Also, theoligonucleotide may comprise a sequence motif which exhibit RNAsecatalytic activity or moieties may be attached to the oligonucleotideswhich when brought into proximity with the target by the hybridizationevent disables the target from further metabolic activities.

It has been shown that β-D-oxy-LNA does not support RNaseH activity.However, this can be changed according to the invention by creatingchimeric oligonucleotides composed of β-D-oxy-LNA and DNA, calledgapmers. A gapmer is based on a central stretch of 4-12 nt DNA ormodified monomers recognizable and cleavable by the RNaseH (the gap)typically flanked by 1 to 6 residues of J-D-oxy-LNA (the flanks). Theflanks can also be constructed with LNA derivatives. There are otherchimeric constructs according to the invention that are able to act viaan RNaseH mediated mechanism. A headmer is defined by a contiguousstretch of β-D-oxy-LNA or LNA derivatives at the 5′-end followed by acontiguous stretch of DNA or modified monomers recognizable andcleavable by the RNaseH towards the 3′-end, and a tailmer is defined bya contiguous stretch of DNA or modified monomers recognizable andcleavable by the RNaseH at the 5′-end followed by a contiguous stretchof f-D-oxy-LNA or LNA derivatives towards the 3′-end. Other chimerasaccording to the invention, called mixmers consisting of an alternatecomposition of DNA or modified monomers recognizable and cleavable byRNaseH and J3-D-oxy-LNA and/or LNA derivatives might also be able tomediate RNaseH binding and cleavage. Since α-L-LNA recruits RNaseHactivity to a certain extent, smaller gaps of DNA or modified monomersrecognizable and cleavable by the RNaseH for the gapmer construct mightbe required, and more flexibility in the mixmer construction might beintroduced. FIG. 1 shows an outline of different designs according tothe invention.

Pharmacokinetic Properties

The clinical effectiveness of antisense oligonucleotides depends to asignificant extent on their pharmacokinetics e.g. absorption,distribution, cellular uptake, metabolism and excretion. In turn theseparameters are guided significantly by the underlying chemistry and thesize and three-dimensional structure of the oligonucleotide.

As mentioned earlier LNA according to the invention is not a single, butseveral related chemistries, which although molecularly different allexhibit stunning affinity towards complementary DNA and RNA. Thus, theLNA family of chemistries are uniquely suited of development oligosaccording to the invention with tailored pharmacokinetic propertiesexploiting either the high affinity of LNA to modulate the size of theactive compounds or exploiting different LNA chemistries to modulate theexact molecular composition of the active compounds. In the latter case,the use of for instance amino-LNA rather than oxy-LNA will change theoverall charge of the oligo and affect uptake and distribution behavior.Likewise the use of thio-LNA instead of oxy-LNA will increase thelipophilicity of the oligonucleotide and thus influence its ability topass through lipophilic barriers such as for instance the cell membrane.

Modulating the pharmacokinetic properties of an LNA oligonucleotideaccording to the invention may further be achieved through attachment ofa variety of different moieties. For instance, the ability ofoligonucleotides to pass the cell membrane may be enhanced by attachingfor instance lipid moieties such as a cholesterol moiety, a thioether,an aliphatic chain, a phospholipid or a polyamine to theoligonucleotide. Likewise, uptake of LNA oligonucleotides into cells maybe enhanced by conjugating moieties to the oligonucleotide thatinteracts with molecules in the membrane, which mediates transport intothe cytoplasm.

Pharmacodynamic Properties

The pharmacodynamic properties can according to the invention beenhanced with groups that improve oligomer uptake, enhance biostabilitysuch as enhanced oligomer resistance to degradation, and/or increase thespecificity and affinity of oligonucleotides hybridisationcharacteristics with target sequence e.g. a mRNA sequence.

Toxicology

There are basically two types of toxicity associated with antisenseoligos:

sequence-dependant toxicity, involving the base sequence, andsequence-independent, class-related toxicity. With the exception of theissues related to immunostimulation by native CpG sequence motifs, thetoxicities that have been the most prominent in the development ofantisense oligonucleotides are independent of the sequence, e.g. relatedto the chemistry of the oligonucleotide and dose, mode, frequency andduration of administration. The phosphorothioates class ofoligonucleotides have been particularly well characterized and found toelicit a number of adverse effects such as complement activation,prolonged PTT (partial thromboplastin time), thrombocytopenia,hepatotoxicity (elevation of liver enzymes), cardiotoxicity,splenomegaly and hyperplasia of reticuloendothelial cells.

As mentioned earlier, the LNA family of chemistries provideunprecedented affinity, very high bio-stablity and the ability tomodulate the exact molecular composition of the oligonucleotide. In oneembodiment of the invention, LNA containing compounds enables thedevelopment of oligonucleotides which combine high potency withlittle—if any—phosphorothioate linkages and which are therefore likelyto display better efficacy and safety than contemporary antisensecompounds.

Manufacture

Oligo- and polynucleotides of the invention may be produced using thepolymerisation techniques of nucleic acid chemistry well known to aperson of ordinary skill in the art of organic chemistry. Generally,standard oligomerisation cycles of the phosphoramidite approach (S. L.Beaucage and R. P. Iyer, Tetrahedron, 1993, 49, 6123; S. L. Beaucage andR. P. Iyer, Tetrahedron, 1992, 48, 2223) is used, but e.g. H-phosphonatechemistry, phosphortriester chemistry can also be used.

For some monomers of the invention longer coupling time, and/or repeatedcouplings with fresh reagents, and/or use of more concentrated couplingreagents were used.

The phosphoramidites employed coupled with satisfactory >98% step-wisecoupling yields. Thiolation of the phosphate is performed by exchangingthe normal, e.g. iodine/pyridine/H₂O, oxidation used for synthesis ofphosphordiester oligomers with an oxidation using Beaucage's reagent(commercially available) other sulfurisation reagents are alsocomprised. The phosphorthioate LNA oligomers were efficientlysynthesised with stepwise coupling yields 98%.

The β-D-amino-LNA, f-D-thio-LNA oligonucleotides, α-L-LNA andβ-D-methylamino-LNA oligonucleotides were also efficiently synthesisedwith step-wise coupling yields 98% using the phosphoramidite procedures.

Purification of LNA oligomeric compounds was done using disposablereversed phase purification cartridges and/or reversed phase HPLC and/orprecipitation from ethanol or butanol. Gel electrophoresis, reversedphase HPLC, MALDI-MS, and ESI-MS was used to verify the purity of thesynthesized oligonucleotides. Furthermore, solid support materialshaving immobilised thereto an optionally nucleobase protected andoptionally 5′-OH protected LNA are especially interesting as materialfor the synthesis of LNA containing oligomeric compounds where an LNAmonomer is included in at the 3′ end. In this instance, the solidsupport material is preferable CPG, e.g. a readily (commercially)available CPG material or polystyrene onto which a 3′-functionalised,optionally nucleobase protected and optionally 5′-OH protected LNA islinked using the conditions stated by the supplier for that particularmaterial.

Indications

The pharmaceutical composition according to the invention can be usedfor the treatment of many different diseases. For example HIF-1α hasbeen found to be overexpressed in various solid human tumours and theirmetastases, e.g. tumours of the breast, colon, prostate, pancreas,brain, lung, ovary, gastro-intestinal system, head and neck, liver,bladder and cervix (Zhong, H. et al., Cancer Research 59, 5830-5835,1999; Talks, K. L. et al., American Journal of Pathology 157(2),411-421, 2000)

The methods of the invention is preferably employed for treatment orprophylaxis against diseases caused by cancer, particularly fortreatment of cancer as may occur in tissue such as lung, breast, colon,prostate, pancreas, liver, brain, testes, stomach, intestine, bowel,spinal cord, sinuses, cervix, urinary tract or ovaries cancer.

Furthermore, the invention described herein encompasses a method ofpreventing or treating cancer comprising a therapeutically effectiveamount of a HIF-1α modulating oligomeric compound, including but notlimited to high doses of the oligomer, to a human in need of suchtherapy. The invention further encompasses the use of a short period ofadministration of a HIF-1α modulating oligomeric compound. Normal,non-cancerous cells divide at a frequency characteristic for theparticular cell type. When a cell has been transformed into a cancerousstate, uncontrolled cell proliferation and reduced cell death results,and therefore, promiscuous cell division or cell growth is a hallmark ofa cancerous cell type. Examples of types of cancer, include, but are notlimited to, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia (e.g.,acute leukemia such as acute lymphocytic leukemia, acute myelocyticleukemia, chronic myeloid leukemia, chronic lymphocytic leukemia,multiple myeloma), colon carcinoma, rectal carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, renal cell carcinoma,hepatoma, bile duct carcinoma, choriocarcinoma, cervical cancer,testicular cancer, lung carcinoma, bladder carcinoma, melanoma, head andneck cancer, brain cancer, cancers of unknown primary site, neoplasms,cancers of the peripheral nervous system, cancers of the central nervoussystem, tumors (e.g., fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,medullary carcinoma, bronchogenic carcinoma, seminoma, embryonalcarcinoma, Wilms' tumor, small cell lung carcinoma, epithelialcarcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, meningioma, neuroblastoma, and retinoblastoma), heavychain disease, metastases, or any disease or disorder characterized byuncontrolled or abnormal cell growth.

Pharmaceutical Composition

It should be understood that the invention also relates to apharmaceutical composition, which comprises a least one antisenseoligonucleotide construct of the invention as an active ingredient. Itshould be understood that the pharmaceutical composition according tothe invention optionally comprises a pharmaceutical carrier, and thatthe pharmaceutical composition optionally comprises further antisensecompounds, chemotherapeutic compounds, anti-inflammatory compounds,antiviral compounds and/or immuno-modulating compounds.

Salts

The oligomeric compound comprised in this invention can be employed in avariety of pharmaceutically acceptable salts. As used herein, the termrefers to salts that retain the desired biological activity of theherein identified compounds and exhibit minimal undesired toxicologicaleffects. Non-limiting examples of such salts can be formed with organicamino acid and base addition salts formed with metal cations such aszinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt,nickel, cadmium, sodium, potassium, and the like, or with a cationformed from ammonia, N,N-dibenzylethylene-diamine, D-glucosamine,tetraethylammonium, or ethylenediamine; or (c) combinations of (a) and(b); e.g., a zinc tannate salt or the like.

Prodrugs

In one embodiment of the invention the oligomeric compound may be in theform of a pro-drug. Oligonucleotides are by virtue negatively chargedions. Due to the lipophilic nature of cell membranes the cellular uptakeof oligonucleotides are reduced compared to neutral or lipophilicequivalents. This polarity “hindrance” can be avoided by using thepro-drug approach (see e.g. Crooke, R. M. (1998) in Crooke, S. T.Antisense research and Application. Springer-Verlag, Berlin, Germany,vol. 131, pp. 103-140). In this approach the oligonucleotides areprepared in a protected manner so that the oligo is neutral when it isadministered. These protection groups are designed in such a way that sothey can be removed then the oligo is taken up be the cells. Examples ofsuch protection groups are S-acetylthioethyl (SATE) orS-pivaloylthioethyl (t-butyl-SATE). These protection groups are nucleaseresistant and are selectively removed intracellulary.

Conjugates

In one embodiment of the invention the oligomeric compound is linked toligands/conjugates. It is way to increase the cellular uptake ofantisense oligonucleotides. This conjugation can take place at theterminal positions 5′/3′-OH but the ligands may also take place at thesugars and/or the bases. In particular, the growth factor to which theantisense oligonucleotide may be conjugated, may comprise transferrin orfolate. Transferrin-polylysine-oligonucleotide complexes orfolate-polylysine-oligonucleotide complexes may be prepared for uptakeby cells expressing high levels of transferrin or folate receptor. Otherexamples of conjugates/lingands are cholesterol moieties, duplexintercalators such as acridine, poly-L-lysine, “end-capping” with one ormore nuclease-resistant linkage groups such as phosphoromonothioate, andthe like.

The preparation of transferrin complexes as carriers of oligonucleotideuptake into cells is described by Wagner et al., Proc. Natl. Acad. Sci.USA 87, 3410-3414 (1990). Cellular delivery of folate-macromoleculeconjugates via folate receptor endocytosis, including delivery of anantisense oligonucleotide, is described by Low et al., U.S. Pat. No.5,108,921. Also see, Leamon et al., Proc. Natl. Acad. Sci. 88, 5572(1991).

Formulations

The invention also includes the formulation of one or moreoligonucleotide compound as disclosed herein. Pharmaceuticallyacceptable binding agents and adjuvants may comprise part of theformulated drug. Capsules, tablets and pills etc. may contain forexample the following compounds: microcrystalline cellulose, gum orgelatin as binders; starch or lactose as excipients; stearates aslubricants; various sweetening or flavouring agents. For capsules thedosage unit may contain a liquid carrier like fatty oils. Likewisecoatings of sugar or enteric agents may be part of the dosage unit. Theoligonucleotide formulations may also be emulsions of the activepharmaceutical ingredients and a lipid forming a micellar emulsion.

An oligonucleotide of the invention may be mixed with any material thatdo not impair the desired action, or with material that supplement thedesired action. These could include other drugs including othernucleoside compounds.

For parenteral, subcutaneous, intradermal or topical administration theformulation may include a sterile diluent, buffers, regulators oftonicity and antibacterials. The active compound may be prepared withcarriers that protect against degradation or immediate elimination fromthe body, including implants or microcapsules with controlled releaseproperties. For intravenous administration the preferred carriers arephysiological saline or phosphate buffered saline.

Preferably, an oligomeric compound is included in a unit formulationsuch as in a pharmaceutically acceptable carrier or diluent in an amountsufficient to deliver to a patient a therapeutically effective amountwithout causing serious side effects in the treated patient.

Administration

The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be (a) oral (b) pulmonary, e.g., by inhalation orinsufflation of powders or aerosols, including by nebulizer;intratracheal, intranasal, (c) topical including epidermal, transdermal,ophthalmic and to mucous membranes including vaginal and rectaldelivery; or (d) parenteral including intravenous, intraarterial,subcutaneous, intraperitoneal or intramuscular injection or infusion; orintracranial, e.g., intrathecal or intraventricular, administration. Inone embodiment the active oligo is administered IV, IP, orally,topically or as a bolus injection or administered directly in to thetarget organ.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, sprays, suppositories, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful. Preferred topical formulations include those inwhich the oligonucleotides of the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Compositionsand formulations for oral administration include but is not restrictedto powders or granules, microparticulates, nanoparticulates, suspensionsor solutions in water or non-aqueous media, capsules, gel capsules,sachets, tablets or minitablets. Compositions and formulations forparenteral, intrathecal or intraventricular administration may includesterile aqueous solutions which may also contain buffers, diluents andother suitable additives such as, but not limited to, penetrationenhancers, carrier compounds and other pharmaceutically acceptablecarriers or excipients.

Delivery

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

Delivery of drug to tumour tissue may be enhanced by carrier-mediateddelivery including, but not limited to, cationic liposomes,cyclodextrins, porphyrin derivatives, branched chain dendrimers,polyethylenimine polymers, nanoparticles and microspheres (Dass C R. JPharm Pharmacol 2002; 54(1)₃₋₂₇).

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels and suppositories. Thecompositions of the present invention may also be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionsmay further contain substances which increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Combination Drug

Oligonucleotides of the invention may be used to abolish the effects ofHIF-1α induction by acute hypoxia induced by androgen withdrawal therapyin prostate cancer.

Oligonucleotides of the invention may also be conjugated to active drugsubstances, for example, aspirin, ibuprofen, a sulfa drug, anantidiabetic, an antibacterial or an antibiotic.

LNA containing oligomeric compounds are useful for a number oftherapeutic applications as indicated above. In general, therapeuticmethods of the invention include administration of a therapeuticallyeffective amount of an LNA-modified oligonucleotide to a mammal,particularly a human.

In a certain embodiment, the present invention provides pharmaceuticalcompositions containing (a) one or more antisense compounds and (b) oneor more other chemotherapeutic agents which function by a non-antisensemechanism. When used with the compounds of the invention, suchchemotherapeutic agents may be used individually (e.g. mithramycin andoligonucleotide), sequentially (e.g. mithramycin and oligonucleotide fora period of time followed by another agent and oligonucleotide), or incombination with one or more other such chemotherapeutic agents or incombination with radiotherapy. All chemotherapeutic agents known to aperson skilled in the art are here incorporated as combinationtreatments with compound according to the invention.

Anti-inflammatory drugs, including but not limited to nonsteroidalanti-inflammatory drugs and corticosteroids, antiviral drags, andimmuno-modulating drugs may also be combined in compositions of theinvention. Two or more combined compounds may be used together orsequentially.

In another embodiment, compositions of the invention may contain one ormore antisense compounds, particularly oligonucleotides, targeted to afirst nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. Two or more combined compoundsmay be used together or sequentially.

Dosage

Dosing is dependent on severity and responsiveness of the disease stateto be treated, and the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.

Optimum dosages may vary depending on the relative potency of individualoligonucleotides. Generally it can be estimated based on EC50s found tobe effective in in vitro and in vivo animal models. In general, dosageis from 0.01 μg to 1 g per kg of body weight, and may be given once ormore daily, weekly, monthly or yearly, or even once every 2 to 10 yearsor by continuous infusion for hours up to several months. The repetitionrates for dosing can be estimated based on measured residence times andconcentrations of the drug in bodily fluids or tissues. Followingsuccessful treatment, it may be desirable to have the patient undergomaintenance therapy to prevent the recurrence of the disease state.

Uses

The LNA containing oligomeric compounds of the present invention can beutilized for as research reagents for diagnostics, therapeutics andprophylaxis. In research, the antisense oligonucleotides may be used tospecifically inhibit the synthesis of HIF-1α genes in cells andexperimental animals thereby facilitating functional analysis of thetarget or an appraisal of its usefulness as a target for therapeuticintervention. In diagnostics the antisense oligonucleotides may be usedto detect and quantitate HIF-1α expression in cell and tissues byNorthern blotting, in-situ hybridisation or similar techniques. Fortherapeutics, an animal or a human, suspected of having a disease ordisorder, which can be treated by modulating the expression of HIF-1α istreated by administering antisense compounds in accordance with thisinvention. Further provided are methods of treating an animal particularmouse and rat and treating a human, suspected of having or being proneto a disease or condition, associated with expression of HIF-1α byadministering a therapeutically or prophylactically effective amount ofone or more of the antisense compounds or compositions of the invention.

Methods

The methods of the invention is preferably employed for treatment orprophylaxis against diseases caused by a disease. One embodiment of theinvention involves a method of inhibiting the expression of HIF-1α, incells or tissues comprising contacting said cells or tissues with thecompound of the invention so that expression of HIF-1α is inhibited.Furthermore, another embodiment is a method of modulating expression ofa gene involved in a disease comprising contacting the gene or RNA fromthe gene with an oligomeric compound wherein said compound has asequence comprising at least an 8 nucleobase portion of SEQ ID NO: 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114 or 115 whereby gene expression is modulated.These compounds may comprise one or more LNA units. The compound mayhybridizes with messenger RNA of the gene to inhibit expression thereof.Another embodiment is a method of treating a mammal suffering from orsusceptible from a cancer disease, comprising, administering to themammal an therapeutically effective amount of an oligonucleotidetargeted to HIF-1α that comprises one or more LNA units. The describedmethods may target a common cancers, as e.g. primary and metastaticbreast, colorectal, prostate, pancreas, other GI-cancers, lung,cervical, ovarian, brain, head and neck, cervix, colon, liver, thyroid,kidney, testes, stomach, intestine, bowel, esophagus, spinal cord,sinuses, bladder or urinary tract tumors, as well as pre-eclampsia,inflammatory bowel disease and Alzheimers disease. The method may alsomodulate angiogenesis as well as red blood cell proliferation, cellularproliferation, iron metabolism, glucose and energy metabolism, pHregulation, tissue invasion, apoptosis, multi-drug resistance, cellularstress response or matrix metabolism comprising contacting a cell withthe antisense compound of claim the invention so that the cell ismodulated.

All documents mentioned herein are incorporated herein by reference.

EXAMPLES

The present invention has been described with specificity in accordancewith certain of its preferred embodiments. Therefore, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

Example 1 Monomer Synthesis

Preparation of the monomers shown in Scheme 2 in which Y and X are —O—and Z and Z* are protected —O— is described in great detail in thereference, Koshkin et al, J. Org. Chem., 2001, 66, 8504-8512; Sørensenet al., J. Am. Chem. Soc., 2002, 124 (10), 2164-2176; Pedersen et al.,Synthesis, 2002, 6, 802-809 and references found therein, where theprotection groups of Z and Z* are respectivelyoxy-N,N-diisopropyl-O-(2-cyanoethyl)phosphoramidite anddimethoxytrityloxy. The preparation of monomers of the Scheme 2 in whichX is —O— and Y is −5- and —N(CH₃)— is described in Rosenbohm, et al.Org. Biomol. Chem., 2003, 1, 655-663.

Example 2 LNA Oligonucleotide Synthesis

All oligonucleotide syntheses are carried out in 1 μmol scale on a MOSSExpedite instrument platform. The synthesis procedures are essentiallycarried out as described in the instrument manual. The LNA monomers usedwere synthesised according Koshin A. A. et al J. Org. Chem., 2001, 66,8504-8512.

Preparation of the LNA Succinyl Hemiester

5′-O-Drat-3′-hydroxy-LNA monomer (500 mg), succinic anhidride (1.2 eq.)and DMAP (1.2 eq.) were dissolved in DCM (35 mL). The reaction wasstirred at room temperature overnight. After extractions with NaH₂PO₄0.1 M pH 5.5 (2×) and brine (1×), the organic layer was further driedwith anhydrous Na₂SO₄ filtered and evaporated. The hemiester derivativewas obtained in 95% yield and was used without any further purification.

Preparation of the LNA-CPG resin

The above prepared hemiester derivative (90 μmol) was dissolved in aminimum amount of DMF, DIEA and pyBOP (90 μmol) were added and mixedtogether for 1 min. This pre-activated mixture was combined withLCAA-CPG (500 Å, 80-120 mesh size, 300 mg) in a manual synthesizer andstirred. After 1.5 h at room temperature, the support was filtered offand washed with DMF, DCM and MeOH. After drying the loading wasdetermined, and resulted to be 57 μmol/g.

Phosphorothioate Cycles

5′-O-Dmt (A(bz), C(bz), G(ibu), and T) linked to CPG (controlled poreglass) were deprotected using a solution of 3% trichloro acetic acid(v/v) in dichloromethane. The resin is washed with acetonitrile.Coupling of phosphoramidites (A(bz), G(ibu), 5-methyl-C(bz)) orT-β-cyanoethylphosphoramidite) is performed by using a solution of 0.08M of the 5′-O-Dmt-protected=Mite in acetonitrile and activation is doneby using DCI (4,5-dicyanoimidazole) in acetonitrile (0.25 M). Couplingis carried out in 2 minutes. Thiolation is carried out by using Beaucagereagent (0.05 M in acetonitrile) and is allowed to react for 3 minutes.The support is thoroughly washed with acetonitrile and the subsequentcapping capping is carried out by using a solution of and aceticanhydride in THF (CAP A) and N-methylimidazole/pyridine/THF (1:1:8) (CAPB) to cap unreacted 5′-hydroxyl groups. The capping step is thenrepeated and the cycle is concluded by acetonitrile washing.

LNA Cycles

5% O-Dmt (locA(bz), locC(bz), locG(ibu) or locT) linked to CPG(controlled pore glass) is deprotected by using the same procedure asabove. Coupling is performed by using 5′-O-Dmt (locA(bz), locC(bz),locG(ibu) or locT)-β-cyanoethylphosphoramidite (0.1 M in acetonitrile)and activation is done by DCI (0.25 M in acetonitrile). Coupling isprolonged to 7 minutes. Capping is done by using acetic anhydride in THF(CAP A) and a solution of N-methylimidazole/pyridine/THF (1:1:8) (CAP B)for 30 sec. The phosphite triester is oxidized to the more stablephosphate triester by using a solution of I₂ and pyridine in THF for 30sec. The support is washed with acetonitrile and the capping step isrepeated. The cycle is concluded by thorough acetonitrile wash.Abbreviations: Dmt: Dimethoxytrityl and DCI: 4,5-Dicyanoimidazole.

Oligonucleotide Cleavage and Deprotection

The oligomers are cleaved from the support and the β-cyanoethylprotecting group removed by treating the support with 35% NH₄OH 1 h atroom temperature. The support is filtered off and the base protectinggroups are removed by raising the temperature to 65° C. for 4 hours. Theoligosolution is then evaporated to dryness.

Oligonucleotide Purification

The oligos are either purified by (reversed-phase) RP-HPLC or (anionexchange) AIE.

RP-HPLC:

Column: VYDAC ™ cat. No. 218TP1010 (vydac) Flow rate: 3 mL/min Buffer:A 0.1M ammonium acetate pH 7.6 B acetonitrile Gradient: Time 0 10 18 2223 28 B % 0 5 30 100 100 0 IE: Column: Resource ™ 15Q (amershampharmacia biotech) Flow rate: 1.2 mL/min Buffer: A 0.1M NaOH B 0.1MNaOH, 2.0M NaCl Gradient: Time 0 1 27 28 32 33 B % 0 25 55 100 100 0

Abbreviations Dmt: Dimethoxytrityl DCI: 4,5-Dicyanoimidazole DMAP:4-Dimethylaminopyridine DCM: Dichloromethane DMF: DimethylformamideTetrahydrofurane DIEA: N,N-diisopropylethylamine

PyBOP: Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium

-   -   hexafluorophosphate

Bz: Benzoyl Ibu: Isobutyryl

Beaucage: 3H-1,2-Benzodithiole-3-one-1,1-dioxideA(bz), C(bz), G(ibu) or T: LNA-monomers (LNA-locked nucleic acid)

Example 3 Cell Culture

Antisense compounds and their effect on target nucleic acid expressioncan be tested in any of a variety of cell types provided that the targetnucleic acid or protein is present at measurable levels. This can beroutinely determined using, for example, RT-PCR or Northern blot orWestern blot analysis. The following cell types are provided forillustrative purposes, but other cell types can be routinely used,provided that the target is expressed in the cell type chosen.

Cell lines were cultured in the appropriate medium as described belowand maintained at 37° C. at 95-98% humidity and 5% CO₂. Cells wereroutinely passaged 2-3 times weekly.

U87-MG:

The human glioblastoma cell line U87-MG was cultured in Modified EagleMedium (MEM) with Earle's salts and 10% Fetal Calf Serum (FCS)

U373:

The human glioblastoma cell line U373 was cultured in Modified EagleMedium (MEM) with Earle's salts and 10% Fetal Calf Serum (FCS)

15PC3:

The human prostate cancer cell line 15PC3 was kindly donated by Dr. F.Baas, Neurozintuigen Laboratory, AMC, The Netherlands and was culturedin DMEM (Sigma)+10% fetal bovine serum (FBS)+Glutamax I+gentamicin

Anaerobic Cell Culture:

To monitor changes in HIF expression under hypoxic conditions, cellswere cultured under anaerobic conditions at an O₂ level of 0.1-1.5% inan incubation bag (Merck) with Anaerocult (Merck) added to chemicallybind O₂. Anaerobic conditions were obtained within 1-2 hours. Cells weresubjected to anoxia for 6 or 18 hours.

Example 4 Treatment with Antisense Oligonucleotide

The cells (described above) were treated with oligonucleotide using thecationic liposome formulation LipofectAMINE 2000 (Gibco) as transfectionvehicle. Cells were seeded in 100 mm×20 mm cell culture petri dishes(Corning) or 6-well plates (NUNC) and treated when 90% confluent. Oligoconcentrations used ranged from 1 nM to 400 nM final concentration.Formulation of oligo-lipid complexes was carried out according to themanufacturers instructions using serum-free MEM and a final lipidconcentration of 5 μg/ml in 6 ml total volume. Cells were incubated at37° C. for 4 or 24 hours and treatment was stopped by removal ofoligo-containing culture medium. Cells were washed and serum-containingMEM was added. After oligo treatment cells were either allowed torecover for 18 hours before they were subjected to anoxia for 6 hours ordirectly subjected to anoxia for 18 hours.

Example 5 Extraction of Total RNA

Total RNA was isolated either using RNeasy mini kit (e.g. Qiagen cat.no. 74104) or using the Trizol reagent (e.g. Life technologies cat. no.15596). For RNA isolation from cell lines, RNeasy is the preferredmethod and for tissue samples Trizol is the preferred method. Total RNAwas isolated from cell lines using the RNeasy mini kit (Qiagen)according to the protocol provided by the manufacturer. Tissue sampleswere homogenised using an Ultra Turrax T8 homogeniser (e.g. IKA Analysentechnik) and total RNA was isolated using the Trizol reagent protocolprovided by the manufacturer.

Example 6 First Strand cDNA Synthesis

First strand synthesis was performed using OmniScript ReverseTranscriptase kit (cat#205113, Qiagen) according to the manufacturersinstructions. For each sample 0.5 μg total RNA was adjusted to 12 iteach with RNase free H₂O and mixed with 2 poly (dT)₁₂₋₁₈ (2.5 μg/ml)(Life Technologies, GibcoBRL, Roskilde, DK), 2 μl dNTP mix (5 mM eachdNTP), 2 μl 10× Buffer RT, 1 μl RNAguard™Rnase INHIBITOR (33.3 U/ml),(cat#27-0816-01, Amersham Pharmacia Biotech, Horshølm, DK) and 1 μlOmniScript Reverse Transcriptase (4 U/μl) followed by incubation at 37°C. for 60 minutes and heat inactivation of the enzyme at 93° C. for 5minutes.

Example 7 Antisense Modulation of HIF-1α Expression Analysis

Antisense modulation of HIF-1α expression can be assayed in a variety ofways known in the art. For example, HIF-1α mRNA levels can bequantitated by, e.g., Northern blot analysis, competitive polymerasechain reaction (PCR), or real-time PCR. Real-time quantitative PCR ispresently preferred. RNA analysis can be performed on total cellular RNAor mRNA.

Methods of RNA isolation and RNA analysis such as Northern blot analysisis routine in the art and is taught in, for example, Current Protocolsin Molecular Biology, John Wiley and Sons.

Real-time quantitative (PCR) can be conveniently accomplished using thecommercially available iQ Multi-Color Real Time PCR Detection System,available from BioRAD.

Real-time Quantitative PCR Analysis of Ha-ras mRNA Levels

Quantitation of mRNA levels was determined by real-time quantitative PCRusing the iQ Multi-Color Real Time PCR. Detection System (BioRAD)according to the manufacturers instructions.

Real-time Quantitative PCR is a technique well known in the art and istaught in for example Heid et al. Real time quantitative PCR, GenomeResearch (1996), 6: 986-994.

Platinum Quantitative PCR SuperMix UDG 2×PCR master mix was obtainedfrom Invitrogen cat#11730. Primers and TaqMan® probes were obtained fromMWG-Biotech AG, Ebersberg, Germany

Probes and primers to human HIF-1α were designed to hybridise to a humanHa-ras sequence, using published sequence information (GenBank accessionnumber NM_(—)001530, incorporated herein as SEQ ID NO:1).

For human HIF-1α the PCR primers were:

forward primer: 5′ CTCATCCAAGAAGCCCTAACGTGTT 3′ (final concentration inthe assay; 0.9 μM) (SEQ ID NO: 116) reverse primer:5′GCTTTCTCTGAGCATTCTGCAAAGC 3′ (final concentration in the assay, 0.9μM) (SEQ ID NO: 117) and the PCR probe was: 5′FAMCCTCAGGAACTGTAGTTCTTTGACTCAAAGCGACATAMRA 3′ (final concentration inthe assay; 0.1 μM) (SEQ ID NO: 118)

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA quantity was usedas an endogenous control for normalizing any variance in samplepreparation. The sample content of human GAPDH mRNA was quantified usingthe human GAPDH ABI Prism Pre-Developed TaqMan Assay Reagent (AppliedBiosystems cat. no. 4310884E) according to the manufacturersinstructions.

For quantification of mouse GAPDH mRNA the following primers and probeswere designed: Sense primer 5′ aaggctgtgggcaaggtcatc 3′ (0.3 μM finalconcentration), antisense primer 5′ gtcagatccacgacggacacatt (0.6 μMfinal concentration), TaqMan probe 5′FAM-gaagctcactggcatggcatggcatccgtgttc-TAMRA 3′ (0.2 μM finalconcentration).

Real Time PCR

The cDNA from the first strand synthesis performed as described inexample 8 was diluted 2-20 times, and analyzed by real time quantitativePCR. The primers and probe were mixed with 2× Platinum Quantitative PCRSuperMix UDG (cat. #11730, Invitrogen) and added to 3.3 μA cDNA to afinal volume of 25 μl. Each sample was analysed in triplicates. Assaying2 fold dilutions of a cDNA that had been prepared on material purifiedfrom a cell line expressing the RNA of interest generated standardcurves for the assays. Sterile H₂O was used instead of cDNA for the notemplate control. PCR program: 50° C. for 2 minutes, 95° C. for 10minutes followed by 40 cycles of 95° C., 15 seconds, 60° C., 1 minutes.

Relative quantities of target mRNA sequence were determined from thecalculated Threshold cycle using the iCycler iQ Real-time DetectionSystem software.

Example 8 Western Blot Analysis of HIF-1α Protein Levels

Protein levels of HIF-1α can be quantitated in a variety of ways wellknown in the art, such as immunoprecipitation, Western blot analysis(immunoblotting), ELISA, RIA (Radio Immuno Assay) orfluorescence-activated cell sorting (FACS). Antibodies directed toHIF-1α can be identified and obtained from a variety of sources, such asUpstate Biotechnologies (Lake Placid, USA), Novus Biologicals(Littleton, Colo.), Santa Cruz Biotechnology (Santa Cruz, Calif.) or canbe prepared via conventional antibody generation methods.

To measure the effect of treatment with antisense oligonucleotidesagainst HIF-1α, protein levels of HIF1-α in treated and untreated cellswere determined using Western blotting.

After treatment with oligonucleotide as described above, cell wereharvested by scraping with a rubber policeman in ice-coldphosphate-buffered saline (PBS) containing 0.37 mg/ml of the proteaseinhibitor phenyl methyl sulfonyl fluoride (PMSF).

The harvested cells were washed in 1 mL PBS containing PMSF as describedabove and cell pellets were kept frozen at −80° C.

For protein extraction, frozen cell pellets were dissolved in 3 volumesof ice-cold lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Non-idet P40 (NP-40), 0.1% SDS, 1% (w/v) natrium-deoxycholat, 1 mM dithiothreitol(DTT), Complete protein inhibitor cocktail (Boehringer Mannheim)). Thesamples were sonicated 2-3 times a 5-10 seconds in a Vibra Cell 50sonicator (Sallies & Materials Inc.). The lysate was stored at −80° C.until further use.

Protein concentration of the protein lysate was determined using the BCAProtein Assay Kit (Pierce) as described by the manufacturer.

SDS Gel Electrophoresis:

Protein samples prepared as described above were thawed on ice anddenatured at 70° C. for 10 min.

Samples were loaded on 1.0 mm 10% NuPage Bis-Tris gel (NOVEX) and gelswere run in running buffer, either NuPage MES SDS Running Buffer orNuPage MOPS SDS Running Buffer (both NOVEX) depending on desiredseparation of proteins in an Xcell II Mini-cell electrophoresis module(NOVEX).

In the inner chamber of the electrophoresis module NuPage Antioxidant(NOVEX) was added to the running buffer at a final concentration of 2.5μl/ml. For size reference, SeeBlue Plus2 Prestained Standard(Invitrogen) was loaded on the gel. The electrophoresis was run at 160 Vfor 2 hours.

Semi-Dry Blotting:

After electrophoresis, the separated proteins were transferred to apolyvinyliden difluoride (PVDF) membrane by semi-dry blotting. The gelwas equilibrated in NuPage Transfer Buffer (NOVEX) until blotted. Theblotting procedure was carried out in a Trans-blot SD Semi-Dry transfercell (BioRAD) according to the manufacturers instructions. The membranewas stored at 4° C. until further use.

Immunodetection:

To detect the desired protein, the membrane was incubated with eitherpolyclonal or monoclonal antibodies against the protein. The membranewas blocked in blocking buffer (2.5% skim milk powder and 5% BSAdissolved in TS-buffer (150 mM NaCl, 10 mM Tris.base pH 7.4)) for 1 hourwith agitation. The membrane was then washed 2×15 min. in TS-buffer atroom temperature and incubated over night with primary antibody inTS-Tween20-buffer with 0.1% NaN₃ at 4 C. The following primarymonoclonal antibodies and concentrations/dilutions were used:Mouse-anti-Glut1 (from T. Ploug, The Panum Institute, Copenhagen) 1:20,mouse-anti-HIF-1α (H72320, Transduction Laboratories) 1 μg/ml,mouse-anti-α-tubulin (T-9026, Sigma) 1:10.000. After incubation with theprimary antibody the membrane was washed in TS-Tween20-buffer for 15minutes followed by 2 additional washes of 5 minutes each with agitationat room temperature. Subsequently the membrane was incubated with a1:5000 dilution of the secondary antibody, peroxidase conjugatedpolyclonal goat-anti-mouse-immunoglobulins (PO447, DAKO A/S) for 1 hourat room temperature. The membrane was then washed in TS-Tween20-bufferfor 15 minutes followed by 3 additional of washes 5 minutes each withagitation at room temperature. After the last wash the membrane wasincubated with ECL⁺ Plus (Amersham), for 5 minutes followed by animmediate scanning with a STORM 840 (Molecular Dynamics Inc.). Themembrane was stripped in stripping-buffer (100 mM 2-mercapto-ethanol, 2%SDS, 62.5 mM Tris-base) pH 6.7 by incubation with low agitation for 30minutes at 50 C. After wash in TS-Tween20-buffer 2×10 minutes at roomtemperature, the membrane was dried and sealed in a plastic bag andstored at 4 C. The Protein expression levels were quantified relative toexpression of a housekeeping protein using Image Quant version 5.0software (Molecular Dynamics Inc).

Example 9 Antisense Inhibition of Human HIF-1α Expression byOligonucleotides

In accordance with the present invention, a series of oligonucleotideswere designed to target different regions of the human HIF-1α RNA, usingpublished sequences (GenBank accession number NM_(—)001530, incorporatedherein as SEQ ID NO: 1 and FIG. 7). The oligonucleotides 16 nucleotidesin length are shown in Table 1 and are having a SEQ ID NO. Theoligonucleotides are designed so they are to be particularly potent asantisense oligonucleotides, particularly when synthesised usingartificial nucleotides such as LNA or phosphorothioates etc. “Targetsite” indicates the first nucleotide number on the particular targetsequence to which the oligonucleotide binds. The compounds were analysedfor their effects on HIF-1α protein levels by Western blot analysis asdescribed in other examples herein.

TABLE 1 Inhibition of human HIF-1αprotein levels antisense oligonucleotides SEQ ID NO./ % inhibitionTarget Oligo Sequence Oligo Design Cureon no. 100 nM oligo site 5′-3′5′-3′ SEQ ID NO 2/ 92 234 GCGATGTCTTCACGGCG_(s)C_(s)G_(s)A_(s)t_(s)g_(s)t_(s)c_(s)t_(s)t_(s)c_(s)a_(s)C_(s)G_(s)G_(s)C2651 SEQ ID NO 3/ 94 2256 TGGTGAGGCTGTCCGAT_(s)G_(s)G_(s)T_(s)g_(s)a_(s)g_(s)g_(s)c_(s)t_(s)g_(s)t_(s)C_(s)C_(s)G_(s)A2627 SEQ ID NO 4 251 ATGGTGAATCGGTCCC SEQ ID NO 5 250 TGGTGAATCGGTCCCCSEQ ID NO 6 49 AGGTGGCTTGTCAGGG SEQ ID NO 7 231 ATGTCTTCACGGCGGGSEQ ID NO 8 233 CGATGTCTTCACGGCG SEQ ID NO 9 2017 GGCTTGCGGAACTGCTSEQ ID NO 10 1471 TTGTGTCTCCAGCGGC SEQ ID NO 11 6 CGAAGAGAGTGCTGCCSEQ ID NO 12/ 96 153 AGGCAAGTCCAGAGGTA_(s)G_(s)G_(s)C_(s)a_(s)a_(s)g_(s)t_(s)c_(s)c_(s)a_(s)g_(s)A_(s)G_(s)G_(s)T2654 SEQ ID NO 13 1937 GCTAACATCTCCAAGT SEQ ID NO 14 1965GAAGTCATCATCCATT SEQ ID NO 15 1744 GTGTCTGATCCTGAAT SEQ ID NO 16 1821ATCCACATAAAAACAA SEQ ID NO 17 2050 CTGTAACTGTGCTTTG SEQ ID NO 18 2182TAGGAGATGGAGATGC SEQ ID NO 19 2317 CGTTAGGGCTTCTTGG SEQ ID NO 20 2506TCCAAGAAAGTGATGT SEQ ID NO 21 2621 CCACTTTCATCCATTG SEQ ID NO 22/ 932680 TTCTGCTGCCTTGTATT_(s)T_(s)C_(s)T_(s)g_(s)c_(s)t_(s)g_(s)c_(s)c_(s)t_(s)t_(s)G_(s)T_(s)A_(s)T2655 SEQ ID NO 23/ 93 2783 TTTAGGTAGTGAGCCAT_(s)T_(s)T_(s)A_(s)g_(s)g_(s)t_(s)a_(s)g_(s)t_(s)g_(s)a_(s)G_(s)C_(s)C_(s)A2656 SEQ ID NO 24 2837 GCAGTATTGTAGCCAG SEQ ID NO 25 3067TATTGGCATCTTCTTA SEQ ID NO 26 3100 TGATGAAAGGTTACTG SEQ ID NO 27 3169GGCAAAGCATTATTAT SEQ ID NO 28 3356 AACCATACAGCATTTA SEQ ID NO 29 3360AATAAACCATACAGCA SEQ ID NO 30 3426 TGCCACATACCTTCTA SEQ ID NO 31 3437ATCCAAATAAATGCCA SEQ ID NO 32 3531 CATAAACTTCCACAAC SEQ ID NO 33 170GCGGAGAAGAGAAGGA SEQ ID NO 34 3582 CCAACAGGGTAGGCAG SEQ ID NO 35 3704AATAGCGACAAAGTGC SEQ ID NO 36 3845 AACCACAAAGAGCAAA SEQ ID NO 37 2369TTTAGTTCTTCCTCAG SEQ ID NO 38 2848 ACCAAGTTTGTGCAGT SEQ ID NO 38 2413TTTTTCGCTTTCTCTG SEQ ID NO 40 2919 CAGCATTAAAGAACAT SEQ ID NO 41 2986AAAATGATGCTACTGC SEQ ID NO 42 2720 TGATCCAAAGCTCTGA SEQ ID NO 43 286TCTTTTTCTTGTCGTT SEQ ID NO 44 3032 ATAAACTCCCTAGCCA SEQ ID NO 45 3228GTAACTGCTGGTATTT SEQ ID NO 46 3299 TAACAATTTCATAGGC SEQ ID NO 47 3490GCTGGCAAAGTGACTA SEQ ID NO 48 3610 TTTACAGTCTGCTCAA SEQ ID NO 49 3677CATTGTATTTTGAGCA SEQ ID NO 50 3786 TTTACTGTGACAACTA SEQ ID NO 51 3874AACAAAACAATACAGT SEQ ID NO 52 384 TGGCAACTGATGAGCA SEQ ID NO 53/ 87 479TCACCAGCATCCAGAAT_(s)C_(s)A_(s)C_(s)c_(s)a_(s)g_(s)c_(s)a_(s)t_(s)c_(s)c_(s)A_(s)GsA_(s)A2657 SEQ ID NO 54/ 84 917 ATCAGCACCAAGCAGGA_(s)T_(s)C_(s)A_(s)g_(s)c_(s)a_(s)c_(s)c_(s)a_(s)a_(s)g_(s)C_(s)A_(s)G_(s)G2658 SEQ ID NO 55/ 95 1177 TGGCAAGCATCCTGTAT_(s)G_(s)G_(s)C_(s)a_(s)a_(s)g_(s)c_(s)a_(s)t_(s)c_(s)T_(s)G_(s)T_(s)A2659 SEQ ID NO 56/ 93 1505 TCTGTGTCGTTGCTGCT_(s)C_(s)T_(s)G_(s)t_(s)g_(s)t_(s)c_(s)g_(s)t_(s)t_(s)g_(s)C_(s)T_(s)G_(s)C2660 SEQ ID NO 57 2095 TGGTGGCATTAGCAGT SEQ ID NO 58 2116CATCAGTGGTGGCAGT SEQ ID NO 59/ 86 2223 TGGTGATGATGTGGCAT_(s)G_(s)G_(s)Tsg_(s)a_(s)t_(s)g_(s)a_(s)t_(s)g_(s)t_(s)G_(s)G_(s)C_(s)A2661 SEQ ID NO 60/ 79 2477 TCGTCTGGCTGCTGTAT_(s)C_(s)G_(s)T_(s)c_(s)t_(s)g_(s)g_(s)c_(s)t_(s)g_(s)c_(s)T_(s)G_(s)T_(s)A2662 SEQ ID NO 61/ 85 2553 TTGCTCCATTCCATTCT_(s)T_(s)G_(s)C_(s)t_(s)c_(s)c_(s)a_(s)t_(s)t_(s)c_(s)c_(s)A_(s)T_(s)T_(s)C2663 SEQ ID NO 62 98 AAGCGGGCGGCAATCG SEQ ID NO 63 349 ATTCTTTACTTCGCCGSEQ ID NO 64 412 CAAGATGCGAACTCAC SEQ ID NO 65 516 ATTCATCTGTGCTITCSEQ ID NO 66 574 TGTCACCATCATCTGT SEQ ID NO 67 747 GCTTCGCTGTGTGTTTSEQ ID NO 68 638 TGTCCAGTTAGTTCAA SEQ ID NO 69 700 TGTGTGTAAGCATTTCSEQ ID NO 70 809 GCAGACTTTATGTTCA SEQ ID NO 71 871 GTTGGTTACTGTTGGTSEQ ID NO 72 968 TTGCTATCTAAAGGAA SEQ ID NO 73 1104 ATCAGAGTCCAAAGCASEQ ID NO 74 1057 GTTCTTCTGGCTCATA SEQ ID NO 75 1003 ATITCATATCCAGGCTSEQ ID NO 76 1163 TACTGTCCTGTGGTGA SEQ ID NO 77 1221 TATGACAGTTGCTTGASEQ ID NO 78 1284 AATACCACTCACAACG SEQ ID NO 79 1322 TCTGTTTGTTGAAGGGSEQ ID NO 80 1383 AACTTTGGTGAATAGC SEQ ID NO 81 1440 TAAAGCATCAGGTTCCSEQ ID NO 82 1559 GGGAGCATTACATCAT SEQ ID NO 83 1613 GTGGGTAATGGAGACASEQ ID NO 84 1669 CTTCTTGATTGAGTGC SEQ ID NO 85 1702 GTGACTCTGGATTTGGSEQ ID NO 86 1783 CAGGTGAACTTTGTCT SEQ ID NO 87 1804 ATTCACTGGGACTATTSEQ ID NO 88 1887 TGCTTCTGTGTCTTCA SEQ ID NO 114/ 97 3091GTTACTGCCTTCTTACG_(s)T_(s)T_(s)A_(s)c_(s)t_(s)g_(s)c_(s)c_(s)t_(s)t_(s)c_(s)T_(s)T_(s)A_(s)CCur2652 SEQ /D NO 115/ 90 293 CCGGCGCCCTCCATGGC_(s)G_(s)G_(s)G_(s)c_(s)g_(s)c_(s)c_(s)c_(s)t_(s)c_(s)c_(s)A_(s)T_(s)G_(s)GCur2653

The sequences that demonstrated at least 20% inhibition of HIF-1αexpression in this experiment are preferred (see also FIG. 1-9). Thetarget sites to which these preferred sequences are complementary areherein referred to as “hot spots” and are therefore preferred sites fortargeting by compounds of the present invention.

Example 10 Antisense Inhibition of HIF-1α by Phosphorothioate, LNAContaining Oligonucleotides or Chimeric Oligonucleotides Having at LeastOne LNA Segments and at Least One Phosphorothioate Segment

In accordance with the present invention, a second series of antisenseoligonucleotides were also synthesized (table 2). These series ofcompounds are full-modified phosphorothioate oligonucleotide, fullmodified LNA oligonucleotide or a chimeric oligonucleotides 16nucleotides in length targeting two different sites. The chimericoligonucleotides are a “gapmer” (GM), “headmen” (WM5) or “tailmer” (WM3)composed of a region consisting of phosphoroptioates (P S) which isflanked on one or both side(s) with a LNA segment. These segments arecomposed of oxy-LNA nucleotides. Some the oligonucleotides also had afluorescent colour (FAM) incorporated. Mismatch oligonucleotides werealso designed (MM). All cytosines in oxy-LNA are methylated at the C5position of the nucleobases. The compounds were analysed for theireffect on HIF-1α protein levels by western blotting as described inother examples herein. “Target site” indicates the first nucleotidenumber on the particular target sequence to which the oligonucleotidebinds.

TABLE 2 Inhibition of human HIF-1α protein levels by phosphorothioateoligonucleotides, LNA containing oligonucleotides or chimeric oligonucleotideshaving one or two LNA segment(s) and one phosphorothioate segment (backbonelinkage is P = O unless other indicated. s; P = S linkage,small letters; deoxynucleic acid, capital letters; oxy-LNA;).% inhibition Target Site & Sequence & Design Name SEQ NO 100 nM oligoDesign 5′ - 3′ Cur0805 SEQ ID 89 24  234 FM GCGATGTCTTCACGGC Cur0806SEQ ID 90 21  234 PSg_(s)c_(s)g_(s)a_(s)t_(s)g_(s)t_(s)c_(s)t_(s)t_(s)c_(s)a_(s)c_(s)g_(s)g_(s)cCur0807 SEQ ID 91  234 GMGCGA_(s)t_(s)g_(s)t_(s)c_(s)t_(s)t_(s)c_(s)a_(s)CGGC Cur0808 SEQ ID 92 234 FAM FAM-GCGA_(s)t_(s)g_(s)t_(s)c_(s)t_(s)t_(s)c_(s)a_(s)CGGCCur0809 SEQ ID 93 34  234 WM5GCGATGTC_(s)t_(s)t_(s)c_(s)a_(s)c_(s)g_(s)g_(s)c Cur0810 SEQ ID 94 54 234 WM3 g_(s)c_(s)g_(s)a_(s)t_(s)g_(s)t_(s)c_(s)TTCACGGC Cur0811SEQ ID 95 24 2256 FM TGGTGAGGCTGTCCGA Cur0812 SEQ ID 96 2256 PSt_(s)g_(s)g_(s)t_(s)g_(s)a_(s)g_(s)g_(s)c_(s)t_(s)g_(s)t_(s)c_(s)c_(s)g_(s)aCur0813 SEQ ID 97 86 2256 GMTGGT_(s)g_(s)a_(s)g_(s)g_(s)c_(s)t_(s)g_(s)t_(s)CCGA Cur0814 SEQ ID 982256 FAM FAM-TGGT_(s)g_(s)a_(s)g_(s)g_(s)c_(s)t_(s)g_(s)t_(s)CCGACur0815 SEQ ID 99 2256 WM5TGGTGAGG_(s)c_(s)t_(s)g_(s)t_(s)c_(s)c_(s)g_(s)a Cur0816 SEQ ID 100 372256 WM3 t_(s)g_(s)g_(s)t_(s)g_(s)a_(s)g_(s)g_(s)CTGTCCGA Cur0959SEQ ID 101  234 MM1 GCGAt_(s)c_(s)t_(s)c_(s)t_(s)t_(s)c_(s)a_(s)GGGCCur0960 SEQ ID 102  234 MM2GCGTt_(s)g_(s)t_(s)c_(s)c_(s)t_(s)c_(s)a_(s)CGGC Cur0961 SEQ ID 1032256 MM1 TGGTg_(s)a_(s)g_(s)c_(s)c_(s)t_(s)g_(s)t_(s)CGGA Cur0962SEQ ID 104 2256 MM2 TGCTg_(s)a_(s)g_(s)g_(s)g_(s)t_(s)g_(s)t_(s)CCGACur2627 SEQ ID 3 94 2256 GMT_(s)G_(s)G_(s)T_(s)g_(s)a_(s)g_(s)g_(s)c_(s)t_(s)g_(s)t_(s)C_(s)C_(s)G_(s)ACur2628 SEQ ID 105 95 2256 GMT_(s)G_(s)G_(s)t_(s)g_(s)a_(s)g_(s)g_(s)c_(s)g_(s)g_(s)t_(s)C_(s)C_(s)G_(s)ACur2629 SEQ ID 106 96 2256 GMT_(s)G_(s)G_(s)t_(s)g_(s)a_(s)g_(s)g_(s)c_(s)t_(s)a_(s)t_(s)C_(s)C_(s)G_(s)aCur2630 SEQ ID 107 95 2256 GMT_(s)G_(s)G_(s)T_(s)g_(s)a_(s)g_(s)g_(s)c_(s)t_(s)g_(s)t_(s)C_(s)C_(s)G_(s)aCur2631 SEQ ID 108 95 2256 GMT_(s)G_(s)G_(s)T_(s)G_(s)A_(s)g_(s)g_(s)c_(s)t_(s)g_(s)t_(s)c_(s)c_(s)g_(s)aCur2632 SEQ ID 109 94 2256 GMTGGt_(s)g_(s)g_(s)a_(s)g_(s)g_(s)c_(s)t_(s)g_(s)t_(s)CCGA Cur2633SEQ ID 110 94 2256 GMTGGt_(s)g_(s)a_(s)g_(s)g_(s)c_(s)t_(s)g_(s)t_(s)CCGa Cur2634 SEQ ID 11191 2256 GM TGGTg_(s)a_(s)g_(s)g_(s)c_(s)t_(s)g_(s)t_(s)CCGa Cur2635SEQ ID 112 94 2256 WM5TGGTGAg_(s)g_(s)c_(s)t_(s)g_(s)t_(s)c_(s)c_(s)g_(s)a Cur2412 SEQ ID 1132256 GM TGGT_(s)g_(s)a_(s)g_(s)g_(s) ^(m)c_(s)t_(s)g_(s)t_(s)CCGA

As shown in Table 2 and FIGS. 1-6, most of SEQ ID NOs 89-104demonstrated at least 20% inhibition of HIF-1α expression in thisexperiment and are therefore preferred. The target sites to which thesepreferred sequences are complementary are herein referred to as “hotspots” and are therefore preferred sites for targeting by compounds ofthe present invention.

Example 11 In Vivo Efficacy Oligomeric Compounds Targeting HIF-1α

The effect of oligonucleotide treatment on growth of tumour xenograftson nude mice can be measured using different tumour cell lines. Examplesof such cell lines are human tumour cell lines U87 (glioblastoma), U373(glioblastoma), 15PC3 (prostate cancer) and CPH 54A (small cell lungcarcinoma) and murine tumour cell line B16 (melanoma).

Treatment of Subcutaneous Tumour Xenografts on Nude Mice UsingLNA-Containing Oligos.

Tumour cells were implanted subcutaneously and then serially passaged bythree consecutive transplantations. Tumour fragments of 1 mm wereimplanted subcutaneously with a trocar needle in NMRI nude mice.Alternatively, cancer cells typically 10⁶ cells suspended in 300 μlmatrigel (BD Bioscience), were subcutaneously injected into the flanksof NMRI nude mice.

Mice were treated by intra-peritoneal or subcutaneous injection ofoligonucleotide at various doses, maximum dose 5 mg/kg/day or byadministration of up to 5 mg/kg/day for up to 28 days using ALZETosmotic pumps implanted subcutaneously. Individual treatment of the micestarted when tumour volume reached 50 mm³. Treatment with PBS wasinitiated when mean tumour volume of control group reached 50 mm³. Theexperiment was terminated when tumours of any group reached maximumallowed sizes. The tumour sizes of all mice were measured daily bycaliper measurements. The effect of treatment was measured as tumoursize and tumour growth rate. Oligonucleotide treated mice weresacrificed 24 hours after the last oligonucleotide injection.

At the end of treatment period mice were anaesthetised and the tumourswere excised and immediately frozen in liquid nitrogen for targetanalysis.

Results: Mice bearing U373 xenograft tumours were treated with Cur813(SEQ ID NO 97) 5 mg/kg/day, i.p.×1 daily for 7 days or PBS100 μl/10g/day, i.p.×1 daily for 7 days. Five mice were treated in each group.Tumour evaluation was carried out as outlined above. Tumour growthcurves are shown in FIG. 9.

Comparison of Tumour Size (t-Test)

Day P-value 4 0.0477 5 0.0156 6 0.0354 7 0.0461

Kaplan Meier Analysis:

Terminal Event: Tumour Size 150 mm³:

Group No. Censored Events Median survival, days PBS 5 0 5 5 Cur813 5 1 47 Logrank test for equality of survival distributions: P = 0.0138

Treatment of Intracranial Tumor Xenografts on Nude Mice UsingLNA-Containing Oligos.

Tumour cells are implanted intracranially on NMRI nude mice andoligonucleotide treatment is initiated 1 week after implantation. Miceare treated by intra-peritonal or subcutaneous injection ofoligonucleotide at various doses, maximum dose 2 mg/kg or PBS. Thenumber of treatments will depend on the tumour growth rate in thecontrol group. The experiment will be terminated when tumours of anygroup reach maximum allowed sizes or until death ensues in any group.The effect of treatment will be measured as time until chronicneurological impairment. Oligonucleotide treated mice will be killed 24hours after the last oligonucleotide injection.

Example 12 In Vivo Analysis: Inhibition of HIF-1α, Protein Level inHuman Tumour Cells Grown In Vivo Systemic Treatment with AntisenseOligonucleotides

The tumours were homogenised in lysis buffer (20 mM Tris-Cl [pH 7.5]; 2%Triton X-100; 1/100 vol. Protease Inhibitor Cocktail Set 111(Calbiochem); 1/100 vol. Protease Inhibitor Cocktail Set II(Calbiochem)) at 4° C. with the use of a motor-driven tissuehomogeniser. 500 μl lysis buffer is applied per 100 mg tumour tissue.Tumour lysates were centrifuged at 13,000 g for 5 min at 4° C. to removetissue debris. Protein concentrations of the tumour extracts weredetermined using the BCA Protein Assay Reagent Kit (Pierce, Rockford).Western blot analysis of target protein expression was carried out asdescribed in example 8.

The present invention has been described with specificity in accordancewith certain of its preferred embodiments. Therefore, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

1-36. (canceled)
 37. A compound of 16 to 50 nucleotides or nucleotideanalogues, wherein adjacent nucleotides or nucleotide analogues arecovalently linked by an internucleotide linkage, and wherein thecompound modulates HIF-1α expression.
 38. The compound according toclaim 37 which inhibits the expression of HIF-1α in a cell or tissue.39. The compound according to claim 37, which is an antisenseoligonucleotide.
 40. The compound according to claim 39, wherein theantisense oligonucleotide comprises at least one nucleotide analogue.41. The compound according to claim 40, wherein the nucleotide analogueis a Locked Nucleic Acid (LNA) unit.
 42. The compound according to claim41, wherein the LNA unit has the structure:

wherein Z and Z* are independently selected from the group consisting ofan internucleotide linkage, a terminal group and a protecting group, Bconstitutes a natural or non-natural nucleobase, X and Y areindependently selected from —O—, —S—, —N(H)—, —N(R)—, —CH₂—, —CH— (ifpart of a double bond), —CH₂—O—, —CH₂—S—, —CH₂—N(H)—, —CH₂—N(R)—,—CH₂—CH₂—, and —CH₂—CH— (if part of a double bond) in which R^(H) isselected from hydrogen and C₁₋₄-alkyl.
 43. The compound according toclaim 41, wherein the LNA unit has a structure of any of the followingformulas:

wherein X is independently selected from —O—, —S—, —NH—, and N(R^(H)) inwhich R^(H) is selected from hydrogen and C₁₋₄-alkyl, Z and Z* areindependently selected from the group consisting of an internucleotidelinkage, a terminal group and a protecting group; and B constitutes anatural or non-natural nucleobase.
 44. The compound according to claim42, wherein the internucleotide linkage is selected from the groupconsisting of —O—P(O)₂—O—, —O—P(O,S)—O—, —O—P(S)₂—O—, —S—P(O)₂—O—,—S—P(O,S)—O—, —S—P(S)₂—O—, —O—P(O)₂—S—, —O—P(O,S)—S—, —S—P(O)₂—S—,—O—PO(R^(H))—O—, O—PO(OCH₃)—O—, —O—PO(NR^(H))—O—, —O—PO(OCH₂CH₂S—R)—O—,—O—PO(BH₃)—O—, —O—PO(NHR^(H))—O—, —O—P(O)₂—NR^(H)—, —NR^(H)—P(O)₂—O—,and —NR″-CO—O—, where R^(H) is selected form hydrogen and C₁₋₄-alkyl.45. The compound of claim 44, wherein at least one internucleotidelinkage is a phosphorothioate linkage.
 46. The compound according toclaim 42, wherein at least one nucleobase is a modified nucleobaseindependently selected from the group consisting of 5-methylcytosine,isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil,6-aminopurine, 2-aminopurine, inosine, diaminopurine, and2-chloro-6-aminopurine.
 47. The compound according to claim 41, whereineach of the LNA units is independently selected from β-D-oxy-LNA,β-D-thio-LNA, β-D-amino-LNA, α-L-oxy-LNA, α-L- thio-LNA andα-L-amino-LNA.
 48. The compound of claim 39, wherein the antisenseoligonucleotide is a chimeric oligonucleotide.
 49. The compound of claim48, wherein the chimeric oligonucleotide is a gapmer, a headmer, or atailmer.
 50. The compound according to claim 41, which comprises atleast four LNA units.
 51. A composition comprising the compound of claim37 and a pharmaceutically acceptable carrier or diluent.
 52. Apharmaceutical composition comprising the compound of claim 37 in theform of a pharmaceutically acceptable salt.
 53. A pharmaceuticalcomposition comprising the compound of claim 37, which is in the form ofa conjugate.
 54. A pharmaceutical composition comprising the compound ofclaim 37, which is in the form of a pro-drug.
 55. A compound representedby the formula:T_(s)G_(s)G_(s)T_(s)g_(s)a_(s)g_(s)g_(s)c_(s)t_(s)g_(s)t_(s)C_(s)C_(s)G_(s)A(SEQ ID NO. 3) wherein uppercase letters denote oxy-LNA nucleotides,lowercase letters denote deoxynucleotides and the subscript “s” denotesa phosphorothioate linkage.
 56. A compound selected from: (SEQ ID NO. 3)T_(s)G_(s)G_(s)T_(s)g_(s)a_(s)g_(s)g_(s)c_(s)t_(s)g_(s)t_(s) ^(Me)C_(s)^(Me)C_(s)G_(s)A, (SEQ ID NO. 96)t_(s)g_(s)g_(s)t_(s)g_(s)a_(s)g_(s)g_(s)c_(s)t_(s)g_(s)t_(s)c_(s)c_(s)g_(s)a,(SEQ ID NO. 97) TGGT_(s)g_(s)a_(s)g_(s)g_(s)c_(s)t_(s)g_(s)t_(s)^(Me)C^(Me)CGA, (SEQ ID NO. 99)TGGTGAGG_(s)c_(s)t_(s)g_(s)t_(s)c_(s)c_(s)g_(s)a (SEQ ID NO. 100)t_(s)g_(s)g_(s)t_(s)g_(s)a_(s)g_(s)g_(s) ^(Me)CTGT^(Me)C^(Me)CGA(SEQ ID NO. 105)T_(s)G_(s)G_(s)t_(s)g_(s)a_(s)g_(s)g_(s)c_(s)t_(s)g_(s)t_(s) ^(Me)C_(s)^(Me)C_(s)G_(s)A, (SEQ ID NO. 106)T_(s)G_(s)G_(s)t_(s)g_(s)a_(s)g_(s)g_(s)c_(s)t_(s)g_(s)t_(s) ^(Me)C_(s)^(Me)C_(s)G_(s)a, (SEQ ID NO. 107)T_(s)G_(s)G_(s)T_(s)g_(s)a_(s)g_(s)g_(s)c_(s)t_(s)g_(s)t_(s) ^(Me)C_(s)^(Me)C_(s)G_(s)a, (SEQ ID NO. 108)T_(s)G_(s)G_(s)T_(s)G_(s)A_(s)g_(s)g_(s)c_(s)t_(s)g_(s)t_(s)c_(s)c_(s)g_(s)a,(SEQ ID NO. 109) TGGt_(s)g_(s)a_(s)g_(s)g_(s)c_(s)t_(s)g_(s)t_(s)^(Me)C^(Me)CGA, (SEQ ID NO. 110)TGGt_(s)g_(s)a_(s)g_(s)g_(s)c_(s)t_(s)g_(s)t_(s) ^(Me)C^(Me)CGa,(SEQ ID NO. 111) TGGTg_(s)a_(s)g_(s)g_(s)c_(s)t_(s)g_(s)t_(s)^(Me)C^(Me)CGa, and (SEQ ID NO. 112)TGGTGAg_(s)g_(s)c_(s)t_(s)g_(s)t_(s)c_(s)c_(s)g_(s)a,

wherein uppercase letters denote oxy-LNA nucleotides, lowercase lettersdenote deoxynucleotides, the subscript “s” denotes a phosphorothioatelinkage, and wherein ^(Me)C denotes an oxy LNA monomer containing a5-methyl cytosine base.